Polypeptide variants

ABSTRACT

The present invention relates to polypeptide variants and methods for obtaining variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/316,393filed Jan. 9, 2019 which is a 35 U.S.C. 371 national application ofPCT/EP2017/067572 filed Jul. 12, 2017, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 2016 00420filed Jul. 13, 2016, the contents of which are fully incorporated hereinby reference.

REFERENCE TO A JOINT RESEARCH AGREEMENT

The embodiments claimed in the present application were made under ajoint research agreement between The Procter & Gamble Company andNovozymes A/S.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel DNase variants exhibitingalterations relative to the parent DNase in one or more propertiesincluding: wash performance, detergent stability and/or storagestability. The variants of the invention are suitable for use incleaning processes and detergent compositions, such as laundrycompositions and dish wash compositions, including hand wash andautomatic dish wash compositions. The present invention also relates toisolated DNA sequences encoding the variants, expression vectors, hostcells, and methods for producing and using the DNase variants of theinvention.

Description of the Related Art

To save energy and protect the environment the temperature is loweredfor many cleaning processes. In addition, many harsh detergentcomponents such as strong bleaches have been replaced, and shortcleaning processes are becoming increasingly more popular. When thetemperature is lowered, bleaches are replaced and when cleaning and washprocesses are shortened, microorganisms such as bacteria have betterconditions for growth. Microorganisms generally live attached tosurfaces in many natural, industrial, and medical environments,encapsulated by extracellular substances including biopolymers andmacromolecules. The resulting layer of slime encapsulated microorganismis termed a biofilm which often are surrounded by a matrix ofextracellular polymeric substance (EPS). EPS is a polymericconglomeration generally composed of extracellular DNA, proteins, andpolysaccharides

The presence of organic matter such as biofilm and EPS may imply thatthe laundry items become sticky and soil may adhere to the sticky areas.When dirty laundry items are washed together with less dirty laundryitems the dirt present in the wash liquor tend to adhere to the laundry(e.g. by re-deposition) in particular if the laundry is sticky asdescribed above. As a result, hereof the laundry item is more “soiled”after wash than before wash.

International patent application WO 2011/098579 (Newcastle UNIV.) and WO2014/087011 (Novozymes A/S) relates to the use of deoxyribonucleasecompounds. To be useful in cleaning processes such as laundry an enzymesuch a DNase need to be stable in detergent compositions and compatiblewith standard detergent components such as surfactants, builders,bleaches etc.

The present invention provides such enzymes which are suitable for usein compositions such as cleaning e.g. detergent compositions.

SUMMARY OF THE INVENTION

The present invention relates to a variant of a DNase parent, whereinthe DNase parent belong to the GYS-clade and wherein the parent DNasecomprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) orASXNRSKG (SEQ ID NO: 26).

The invention further relates to a DNase variant comprising analteration compared to the polypeptide shown in SEQ ID NO: 1 at one ormore positions corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10, 12,13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38, 39, 40, 42,49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78, 79, 80, 82,83, 92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112, 116, 125, 126,127, 130, 132, 135, 138, 139, 143, 144, 145, 147, 149, 152, 156, 157,159, 160, 161, 162, 164, 166, 167, 168, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 181 and 182 of SEQ ID NO: 1, wherein the variant hasa sequence identity to the entire length of the polypeptide shown in SEQID NO: 1 of at least 80%, of at least 85%, at least 90%, at least 96%,at least 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide shown in SEQ ID NO: 1, wherein the varianthas DNase activity.

The invention further relates to a variant of a DNase parent, whereinthe variant comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ IDNO: 25) or ASXNRSKG (SEQ ID NO: 26), wherein the variant comprises oneor more substitution(s) compared to SEQ ID NO 1, wherein thesubstitution is selected from the group consisting of: T1I, T1V, T1Y,T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L,S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10V, A10L, A10K, Q12S, Q12V,Q12E, S13D, S13Y, S13Q, S13F, S13R, S13V, S13N, S13H, S13M, S13W, S13K,S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, T19K, T19L, T19S,T19I, T19V, K21E, K21M, T22P, T22A, T22V, T22D, T22R, T22K, T22M, T22E,T22H, T22L, T22W, T22F, T22C, T22I, G24Y, S25P, S27N, S27I, S27M, S27D,S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T,D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V,S42C, S42L, S42M, S42F, S42W, V49R, L51I, K52I, K52H, A55S, D56I, D56L,D56T, S57W, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C,S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L,T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68Y, S68H, S68C, S68T, S68L,V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76F,T77N, T77Y, T77W, T77R, F78I, F78H, F78Y, F78C, T79G, T79R, N80K, S82L,S82E, S82K, S82R, S82H, D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D,S101A, S102M, S102L, S102V, S102A, S102K, S102T, S102R, T104P, T104A,T105V, T105I, K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A,K107D, Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A,S116E, S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, S130E,G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144L, S144P,S144E, S144K, G145V, G145E, G145D, G145A, A147Q, A147W, A147S, G149S,K152H, K152R, S156C, S156G, S156K, S156R, S156T, S156A, T157S, Y159F,K160V, W161L, W161Y, G162Q, G162D, G162M, G162R, G162A, G162S, G162E,G162L, G162K, G162V, G162H, S164R, S164T, Q166D, S167M, S167L, S167F,S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q, S167V, S167T,S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D, T171E,T171A, T171C, A172G, A172S, L173T, L173A, L173V, Q174L, G175D, G175E,G175N, G175R, M176H, L177I, N178D, N178E, N178T, N178S, N178A, S179E,S181R, S181E, S181D, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q,S181G, S181A, Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D,Y182Q, Y182F, Y182L, Y182N, Y182I, Y182E, Y182T and Y182W, wherein thevariant has a sequence identity to the polypeptide shown in SEQ ID NO: 1of at least 80%, at least 85%, at least 90%, at least 96%, at least 97%,at least 98%, or at least 99%, but less than 100% sequence identity tothe polypeptide shown in SEQ ID NO: 1, wherein the variant has DNaseactivity In one aspect the variant has at least one improved propertycompared to the polypeptide shown in SEQ ID NO: 1. In some aspects, theimproved property is increased stability measured as half-lifeimprovement factor, HIF, compared to the DNase having the polypeptideshown in SEQ ID NO: 1.

One aspect of the invention relates to a cleaning e.g. detergentcomposition comprising a variant according to the invention. On aspectrelates to the use of such composition in a cleaning process, such aslaundry or hard surface cleaning such as dish wash.

One aspect relates to a method for obtaining a DNase variant,comprising;

-   -   a) introducing into a parent DNase an alteration at one or more        positions corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10,        12, 13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38,        39, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76,        77, 78, 79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107,        109, 112, 116, 125, 126, 127, 130, 132, 135, 138, 139, 143, 144,        145, 147, 149, 152, 156, 157, 159, 160, 161, 162, 164, 166, 167,        168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 181 and        182 of SEQ ID NO 1, wherein the variant has DNase activity,    -   b) and recovering the variant.

One aspect relates to a method for obtaining a DNase variant comprisingintroducing into a parent DNase an alteration at one or more positions,wherein the alteration is a substitution and wherein the substitution isselected from the group consisting of: T1I, T1L, T1V, T1F, T1Y, T1M,T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F,S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L, A10K, Q12S, Q12V,Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H, S13M, S13W,S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, A17S, T19K,T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P, T22A, T22V, T22D,T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22S, T22I, G24Y,S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A, S27C, S27L,S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P,S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C, S42D, S42L, S42M,S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S, D56I, D56L, D56T,S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C,S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L,T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y, S68H, S68C, S68T,S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R,V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V, F78Y, F78C,T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H, D83C, D83F, D83L,L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L, S102V, S102A, S102K,S102T, S102R, T104S, T104P, T104A, T105V, T105I, K107L, K107C, K107R,K107H, K107S, K107M, K107E, K107A, K107Q, K107D, Q109K, Q109R, Q109S,A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E, S116K, A125K,S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E, G132R, D135R,T138Q, W139R, R143E, R143K, S144Q, S144H, S144A, S144L, S144P, S144E,S144K, G145V, G145E, G145D, G145A, A147H, A147R, A147K, A147Q, A147W,A147N, A147S, G149S, K152H, K152R, S156C, S156G, S156K, S156R, S156T,S156A, T157S, Y159H, Y159F, K160R, K160V, W161L, W161Y, G162Q, G162N,G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K, G162V, G162H,S164R, S164H, S164N, S164T, Q166D, S167M, S167L, S167F, S167W, S167E,S167A, S167Y, S167H, S167C, S167I, S167Q, S167V, S167T, S168V, S168E,S168D, S168L, K170S, K170L, K170F, K170R, T171D, T171E, T171N, T171A,T171S, T171C, A172G, A172S, L173T, L173A, L173V, Q174L, G175D, G175E,G175N, G175R, G175S, M176H, L177I, N178D, N178E, N178T, N178S, N178A,S179E, S181R, S181E, S181D, S181I, S181F, S181H, S181W, S181L, S181M,S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C, Y182K, Y182G, Y182A,Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I, Y182E, Y182T andY182W compared to the polypeptide shown in SEQ ID NO: 1.

In one aspect, the DNase parent belong to the GYS-clade and wherein theparent DNase comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ IDNO: 25) or ASXNRSKG (SEQ ID NO: 26).

One aspect of the invention relates to the use of a DNase variantaccording to the invention for deep cleaning of an item, wherein theitem is a textile or a hard surface.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.The invention further relates to detergent compositions comprising suchvariants.

Sequences

SEQ ID NO: 1 mature polypeptide obtained from Bacillus cibi

SEQ ID NO: 2 mature polypeptide obtained from Bacillus horikoshii

SEQ ID NO: 3 mature polypeptide obtained from Bacillus sp-62520

SEQ ID NO: 4 mature polypeptide obtained from Bacillus horikoshii

SEQ ID NO: 5 mature polypeptide obtained from Bacillus horikoshii

SEQ ID NO: 6 mature polypeptide obtained from Bacillus sp-16840

SEQ ID NO: 7 mature polypeptide obtained from Bacillus sp-16840

SEQ ID NO: 8 mature polypeptide obtained from Bacillus sp-62668

SEQ ID NO: 9 mature polypeptide obtained from Bacillus sp-13395

SEQ ID NO: 10 mature polypeptide obtained from Bacillus horneckiae

SEQ ID NO: 11 mature polypeptide obtained from Bacillus sp-11238

SEQ ID NO: 12 mature polypeptide obtained from Bacillus sp-62451

SEQ ID NO: 13 mature polypeptide obtained from Bacillus sp-18318

SEQ ID NO: 14 mature polypeptide obtained from Bacillus idriensis

SEQ ID NO: 15 mature polypeptide obtained from Bacillus algicola

SEQ ID NO: 16 mature polypeptide obtained from environmental sample J

SEQ ID NO: 17 mature polypeptide obtained from Bacillus vietnamensis

SEQ ID NO: 18 mature polypeptide obtained from Bacillus hwajinpoensis

SEQ ID NO: 19 mature polypeptide obtained from Paenibacillusmucilaginosus

SEQ ID NO: 20 mature polypeptide obtained from Bacillus indicus

SEQ ID NO: 21 Mature polypeptide obtained from Bacillus marisflavi

SEQ ID NO: 22 mature polypeptide obtained from Bacillus luciferensis

SEQ ID NO: 23 mature polypeptide obtained from Bacillus marisflavi

SEQ ID NO: 24 mature polypeptide obtained from Bacillus sp. SA2-6

SEQ ID NO: 25 motif [D/M/L][S/T]GYSR[D/N]

SEQ ID NO: 26 motif ASXNRSKG

Definitions

The term “adjunct materials” means any liquid, solid or gaseous materialselected for the particular type of detergent composition desired andthe form of the product (e.g., liquid, granule, powder, bar, paste,spray, tablet, gel, or foam composition), which materials are alsopreferably compatible with the DNase variant enzyme used in thecomposition. In some aspects, granular compositions are in “compact”form, while in other aspects, the liquid compositions are in a“concentrated” form.

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

“Biofilm” may be produced by any group of microorganisms in which cellsstick to each other on a surface, such as a textile, dishware or hardsurface. These adherent cells are frequently embedded within aself-produced matrix of extracellular polymeric substance (EPS). BiofilmEPS is a polymeric conglomeration generally composed of extracellularDNA, proteins, and polysaccharides. Biofilms may form on living ornon-living surfaces. The microbial cells growing in a biofilm arephysiologically distinct from planktonic cells of the same organism,which, by contrast, are single-cells that may float or swim in a liquidmedium. Bacteria living in a biofilm usually have significantlydifferent properties from free-floating bacteria of the same species, asthe dense and protected environment of the film allows them to cooperateand interact in various ways. One benefit of this environment isincreased resistance to detergents and antibiotics, as the denseextracellular matrix and the outer layer of cells protect the interiorof the community. On laundry biofilm producing bacteria can be foundamong the following species: Acinetobacter sp., Aeromicrobium sp.,Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonassp., Staphylococcus epidermidis, and Stenotrophomonas sp.

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aprokaryotic or eukaryotic cell. A cDNA lacks intron sequences that maybe present in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

The term “clade” means a group of polypeptides clustered together basedon homologous features traced to a common ancestor. Polypeptide cladescan be visualized as phylogenetic trees and a clade is a group ofpolypeptides that consists of a common ancestor and all its linealdescendants. Polypeptides forming a group e.g. a clade as shown in aphylogenetic tree may often share common properties and are more closelyrelated than other polypeptides not in the clade.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of its polypeptide product. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

The term “control sequences” means all components necessary for theexpression of a polynucleotide encoding a variant of the presentinvention. Each control sequence may be native or foreign to thepolynucleotide encoding the variant or native or foreign to each other.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers forintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the polynucleotide encodinga variant.

By the term “deep cleaning” is meant reduction or removal of componentsof biofilm, EPS or parts hereof, such as proteins, DNA, polysaccharides,soil or other components present in e.g. the biofilm or EPS.

The term “detergent composition”, includes unless otherwise indicated,granular or powder-form all-purpose or heavy-duty washing agents,especially cleaning detergents; liquid, gel or paste-form all-purposewashing agents, especially the so-called heavy-duty liquid (HDL) types;liquid fine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, including antibacterial hand-wash types, cleaningbars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos,bathroom cleaners; hair shampoos and hair-rinses; shower gels, foambaths; metal cleaners; as well as cleaning auxiliaries such as bleachadditives and “stain-stick” or pre-treat types. The terms “detergentcomposition” and “detergent formulation” are used in reference tomixtures which are intended for use in a wash medium for the cleaning ofsoiled objects. In some aspects, the term is used in reference tolaundering fabrics and/or garments (e.g., “laundry detergents”). Inalternative aspects, the term refers to other detergents, such as thoseused to clean dishes, cutlery, etc. (e.g., “dishwashing detergents”). Itis not intended that the present invention be limited to any particulardetergent formulation or composition. The term “detergent composition”is not intended to be limited to compositions that contain surfactants.It is intended that in addition to the variants according to theinvention, the term encompasses detergents that may contain, e.g.,surfactants, builders, chelators or chelating agents, bleach system orbleach components, polymers, fabric conditioners, foam boosters, sudssuppressors, dyes, perfume, tannish inhibitors, optical brighteners,bactericides, fungicides, soil suspending agents, anti-corrosion agents,enzyme inhibitors or stabilizers, enzyme activators, transferase(s),hydrolytic enzymes, oxido reductases, bluing agents and fluorescentdyes, antioxidants, and solubilizers.

The term “DNase”, “DNase variants” or “DNase parent” means a polypeptidewith DNase activity that catalyzes the hydrolytic cleavage ofphosphodiester linkages in DNA, thus degrading DNA. DNases belong to theesterases (EC-number 3.1), a subgroup of the hydrolases. The DNases areclassified EC 3.1.21.X, where X=1, 2, 3, 4, 5, 6, 7, 8 or 9. The term“DNase” and the expression “a polypeptide with DNase activity” may beused interchangeably throughout the application. For purposes of thepresent invention, DNase activity is determined according to theprocedure described in the Assay I or Assay II. In some aspects, theDNase variants of the present invention have improved DNase activitycompared to the parent DNase. In some aspects, the DNase variants of thepresent invention have at least 100%, e.g., at least 110%, at least120%, at least 130%, at least 140%, at least 150%, at least 160%, atleast 170%, at least 180%, at least 190%, or at least 200% DNaseactivity compared to the polypeptide shown in SEQ ID NO: 1.

The term “effective amount of enzyme” refers to the quantity of enzymenecessary to achieve the enzymatic activity required in the specificapplication, e.g., in a defined detergent composition. Such effectiveamounts are readily ascertained by one of ordinary skill in the art andare based on many factors, such as the particular enzyme used, thecleaning application, the specific composition of the detergentcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, and the like. The term “effective amount” of aDNase variant refers to the quantity of DNase variant describedhereinbefore that achieves a desired level of enzymatic activity, e.g.,in a defined detergent composition.

The term “expression” includes any step involved in the production ofthe variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to additional nucleotides that provide for its expression.

The term “fabric” encompasses any textile material. Thus, it is intendedthat the term encompass garments, as well as fabrics, yarns, fibers,non-woven materials, natural materials, synthetic materials, and anyother textile material.

The term “high detergent concentration” system includes detergentswherein greater than about 2000 ppm of detergent components is presentin the wash water. European detergents are generally considered to behigh detergent concentration systems.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, and the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The term “improved property” means a characteristic associated with avariant that is improved compared to the parent and/or compared to aDNase with SEQ ID NO: 1, or compared to a DNase having the identicalamino acid sequence of said variant but not having the alterations atone or more of said specified positions. Such improved propertiesinclude, but are not limited to, stability, such as detergent stability,wash performance e.g. deep cleaning effect and the deep-cleaning effectmay include but is not limited to de-gluing effect.

The term “improved DNase activity” is defined herein as an altered DNaseactivity e.g. by increased catalyse of hydrolytic cleavage ofphosphodiester linkages in the DNA the DNase variant displaying analteration of the activity relative (or compared) to the activity of theparent DNase, such as compared to a DNase with SEQ ID NO: 1.

The term “low detergent concentration” system includes detergents whereless than about 800 ppm of detergent components is present in the washwater. Asian, e.g., Japanese detergents are typically considered lowdetergent concentration systems.

The term “improved wash performance” includes but is not limited to theterm “deep cleaning effect”. Improved performance e.g. deep cleaningperformance of a DNase variant according to the invention is measuredcompared to the DNase parent e.g. the DNase shown in SEQ ID NO: 1. Theimproved performance e.g. deep cleaning performance may be expressed asa Remission value of the stained swatches. After washing and rinsing theswatches are spread out flat and allowed to air dry at room temperatureovernight. All washed swatches are evaluated the day after the wash.Light reflectance evaluations of the swatches are done using a MacbethColor Eye 7000 reflectance spectrophotometer with very small aperture.The measurements are made without UV in the incident light and remissionat 460 nm was extracted. Positive response indicate that soil has beenremoved including soiled sticked to the fabric due to e.g. biofilmsticky layer.

The term “isolated polynucleotide” means a polynucleotide that ismodified by the hand of man. In some aspects, the isolatedpolynucleotide is at least 1% pure, e.g., at least 5% pure, at least 10%pure, at least 20% pure, at least 40% pure, at least 60% pure, at least80% pure, at least 90% pure, and at least 95% pure, as determined byagarose electrophoresis. The polynucleotides may be of genomic, cDNA,RNA, semisynthetic, synthetic origin, or any combinations thereof.

The term “laundering” relates to both household laundering andindustrial laundering and means the process of treating textiles with asolution containing a cleaning or detergent composition of the presentinvention. The laundering process can for example be carried out usinge.g. a household or an industrial washing machine or can be carried outby hand.

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In some aspects, the mature polypeptide is aminoacids 1 to 182 of SEQ ID NO: 1. The N-terminals of the maturepolypeptide used according to the present invention were experimentallyconfirmed based on EDMAN N-terminal sequencing data and Intact MS data.It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide.

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having DNase activity.

The term “medium detergent concentration” system includes detergentswherein between about 800 ppm and about 2000 ppm of detergent componentsis present in the wash water. North American detergents are generallyconsidered to be medium detergent concentration systems.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic. The termnucleic acid construct is synonymous with the term “expression cassette”when the nucleic acid construct contains the control sequences requiredfor expression of a coding sequence of the present invention.

The term “non-fabric detergent compositions” include non-textile surfacedetergent compositions, including but not limited to compositions forhard surface cleaning, such as dishwashing detergent compositionsincluding manual dish wash compositions, oral detergent compositions,denture detergent compositions, and personal cleansing compositions.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directs theexpression of the coding sequence.

The term “parent” DNase, DNase parent or precursor DNase may be usedinterchangeably. In context of the present invention “parent DNase” isto be understood as a DNase into which at least one alteration is madein the amino acid sequence to produce a DNase variant having an aminoacid sequence which is less than 100% identical to the DNase sequenceinto which the alteration was made i.e. the parent DNase. Thus, theparent is a DNase having identical amino acid sequence compared to thevariant but not having the alterations at one or more of the specifiedpositions. It will be understood, that in the present context theexpression “having identical amino acid sequence” relates to 100%sequence identity. In a particular aspect the DNase parent is a DNasehaving at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, e. g. at least 99.1%, at least 99.2%, at least 99.3%,at least 99.4%, at least 99.5%, at least 99.6 or 100% identity to apolypeptide shown in SEQ ID NO: 1.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,detergent concentration, type of detergent and water hardness, actuallyused in households in a detergent market segment.

The term “stability” includes storage stability and stability duringuse, e.g. during a wash process and reflects the stability of the DNasevariant according to the invention as a function of time e.g. how muchactivity is retained when the DNase variant is kept in solution inparticular in a detergent solution. The stability is influenced by manyfactors e.g. pH, temperature, detergent composition e.g. amount ofbuilder, surfactants etc. The DNase stability may be measured asdescribed in example 2. The term “improved stability” or “increasedstability” is defined herein as a variant DNase displaying an increasedstability in solutions, relative to the stability of the parent DNaseand/or relative to SEQ ID NO: 1. “Improved stability” and “increasedstability” includes detergent stability. The term “detergent stability”or “improved detergent stability” may be improved stability of the DNaseactivity compared to the DNase parent. The DNase stability is measuredas described in example 2.

The term “substantially pure variant” means a preparation that containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated.Preferably, the variant is at least 92% pure, e.g., at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, at least 99.5% pure, and 100% pure by weight of thetotal polypeptide material present in the preparation. The variants ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant by well-knownrecombinant methods or by classical purification methods.

The term “textile” refers to woven fabrics, as well as staple fibers andfilaments suitable for conversion to or use as yarns, woven, knit, andnon-woven fabrics. The term encompasses yarns made from natural, as wellas synthetic (e.g., manufactured) fibers. The term, “textile materials”is a general term for fibers, yarn intermediates, yarn, fabrics, andproducts made from fabrics (e.g., garments and other articles).

The term “transcription promoter” is used for a promoter which is aregion of DNA that facilitates the transcription of a particular gene.Transcription promoters are typically located near the genes theyregulate, on the same strand and upstream (towards the 5′ region of thesense strand).

The term “transcription terminator” is used for a section of the geneticsequence that marks the end of gene or operon on genomic DNA fortranscription.

The term “variant” means a polypeptide having DNase activity and whichcomprises a substitution at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid, a deletion means removal of an amino acidoccupying a position and an insertion means adding amino acids e.g. 1 to10 amino acids, preferably 1-3 amino acids adjacent to an amino acidoccupying a position. The variants of the present invention have atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of the DNaseactivity of the mature polypeptide shown in SEQ ID NO: 1. The term“DNase variant” means a DNase having DNase activity and comprise analteration, i.e., a substitution, insertion, and/or deletion at one ormore (or one or several) positions compared to the parent DNase e.g.compared to SEQ ID NO: 1.

The term “water hardness” or “degree of hardness” or “dH” or “° dH” asused herein refers to German degrees of hardness. One degree is definedas 10 milligrams of calcium oxide per liter of water.

The term “wild-type DNase” means a DNase expressed by a naturallyoccurring organism, such as a fungal, bacterium, archaea, yeast, plantor animal found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the polypeptide disclosed in SEQID NO: 1 is used to determine the corresponding amino acid residue inanother DNase. The amino acid sequence of another DNase is aligned withthe polypeptide disclosed in SEQ ID NO: 1, and based on the alignment,the amino acid position number corresponding to any amino acid residuein the polypeptide disclosed in SEQ ID NO: 1 is determined using e.g.the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.Biol. 48: 443-453) as implemented in the Needle program of the EMBOSSpackage (EMBOSS: The European Molecular Biology Open Software Suite,Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0or later. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in another DNasecan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), usingtheir respective default parameters.

When the another enzyme has diverged from the polypeptide of SEQ ID NO:1 such that traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample, the SCOP super families of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letters amino acid abbreviations are employed.Amino acid positions are indicated with #₁, #₂, etc.

Substitutions: For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of valine at position #₁ withalanine is designated as “Val #₁Ala” or “V #₁A”. Multiple mutations areseparated by addition marks (“+”) or by commas (,), e.g., “Val#₁Ala”+“Pro #₂Gly” or V #₁A, P #₂G, representing substitutions atpositions #₁ and #₂ of valine (V) and proline (P) with alanine (A) andglycine (G), respectively. If more than one amino acid may besubstituted in a given position these are listed in brackets, such as[X] or {X}. Thus, if both Trp and Lys may be substituted instead of theamino acid occupying at position #₁ this is indicated as X #₁ {W, K}, X#₁ [W, K] or X #₁ [W/K], where the X indicate the amino acid residuepresent at the position of the parent DNase e.g. such as a DNase shownin SEQ ID NO: 1 or a DNase having at least 60% identity hereto. In somecases the variants may be represented as #₁ {W, K} or X #₂P indicatingthat the amino acids to be substituted vary depending on the parent. Forconvenience, as SEQ ID NO: 1 is used for numbering the substitutions,the amino acid in the corresponding position in SEQ ID NO: 1 isindicated, e.g. T1A. However, it will be clear to the skilled artisanthat a DNase variant comprising T1A is not limited to parent DNaseshaving Threonine at a position corresponding to position 1 of SEQ IDNO: 1. In a parent DNase having e.g. asparagine in position 1, theskilled person would translate the mutation specified as T1A to N1A. Inthe event the parent DNase has alanine in position 1, the skilled personwould recognize that the parent DNase is not changed at this position.The same applies for deletions and insertions described below.

Deletions: For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofvaline at position #₁ is designated as “Val #₁*” or “V #₁*”. Multipledeletions are separated by addition marks (“+”) or commas, e.g., “Val#₁*+Pro #₂*” or “V #₁*, P #₂*”.

Insertions: The insertion of an additional amino acid residue such ase.g. a lysine after Val #₁ may be indicated by: Val #₁ValLys or V #₁VK.Alternatively, insertion of an additional amino acid residue such aslysine after V #₁ may be indicated by: * #₁aK. When more than one aminoacid residue is inserted, such as e.g. a Lys, and Gly after #₁ this maybe indicated as: Val #₁ValLysGly or V #₁VKG. In such cases, the insertedamino acid residue(s) may also be numbered by the addition of lower caseletters to the position number of the amino acid residue preceding theinserted amino acid residue(s), in this example: * #₁aK* #₁bG.

Multiple alterations: Variants comprising multiple alterations areseparated by addition marks (“+”) or by commas (,), e.g., “Val #₁Trp+Pro#₂Gly” or “V #₁W, P #₂G” representing a substitution of valine andproline at positions #₁ and #₂ with tryptophan and glycine, respectivelyas described above.

Different alterations: Where different alterations can be introduced ata position, the different alterations may be separated by a comma, e.g.,“Val #₁Trp, Lys” or V #₁W, K representing a substitution of valine atposition #₁ with tryptophan or lysine. Thus, “Val #₁Trp, Lys+Pro #₂Asp”designates the following variants: “Val #₁Trp+Pro #₂Asp”, “Val #₁Lys+Pro#₂Asp” or V #₁W, K+P #₂D.

Specific for Nomenclature of Clades

For purposes of the present invention, the nomenclature [IV] or [I/V]means that the amino acid at this position may be isoleucine (Ile, I) orvaline (Val, V). Likewise, the nomenclature [LVI] and [L/V/I] means thatthe amino acid at this position may be a leucine (Leu, L), valine (Val,V) or isoleucine (Ile, I), and so forth for other combinations asdescribed herein. Unless otherwise limited further, the amino acid X isdefined such that it may be any of the 20 natural amino acids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel DNases preferably obtained fromBacillus, in particular, Bacillus cibi. The DNases of the inventioncomprise at least 60% sequence identity to a polypeptide with SEQ ID NO:1 and comprise an alteration of at least one amino acid positioncompared to the DNase with SEQ ID NO: 1. In some aspects, a DNasevariant of the invention comprise an amino acid sequence comprising atleast one substitution of an amino acid made at a position equivalent toa position in SEQ ID NO: 1. In some aspects, the DNase variant comprisean amino acid sequence comprising at least one deletion of an amino acidmade at a position equivalent to a position in SEQ ID NO: 1. In someaspects, the DNase variant comprise an amino acid sequence comprising atleast one insertion of an amino acid made at a position equivalent to aposition in SEQ ID NO: 1. The present invention also relates to methodsfor of producing DNase variants. The invention further relates to ascreening process. It is not intended that the present invention belimited to any particular method of producing variants or screeningprocesses. The DNase variants of the present invention have at least oneimproved property compared to the parent DNase or compared to SEQ IDNO: 1. Properties includes but are not limited to: stability such as;stability in detergents, storage stability, in wash stability and thermostability, wash performance in particular deep-cleaning performance,increased expression level and malodor reduction.

DNase variants

Some aspects of the invention relate to DNase variants of SEQ ID NO: 1or variants of a DNase having at least 60% identity hereto and tomethods for production of a DNase variant of SEQ ID NO: 1 or a DNasehaving at least 60% identity hereto.

The present invention relates to a DNase comprising an alteration at oneor more positions selected from the list consisting of 1, 4, 5, 6, 7, 8,9, 10, 12, 13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38,39, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78,79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112, 116,125, 126, 127, 130, 132, 135, 138, 139, 143, 144, 145, 147, 149, 152,156, 157, 159, 160, 161, 162, 164, 166, 167, 168, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 181 and 182, wherein each positioncorresponds to the position of the polypeptide of SEQ ID NO: 1, whereinthe variant has DNase activity and wherein the variant has at least oneimproved property compared to the DNase having the amino acid sequenceshown in in SEQ ID NO 1.

In some aspects, the variant has sequence identity at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, such as at least 96%, at least 97%, at least98%, or at least 99%, but less than 100%, to the amino acid sequence ofthe mature parent DNase.

In some aspects, the variant has at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, such as at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, sequence identity to the polypeptide shown in in SEQID NO: 1.

The invention further relates to variants of a DNase parent comprisingSEQ ID NO: 1 wherein said variant comprises an alteration compared toSEQ ID NO: 1 in at least one position selected from the positions: 1, 4,5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29,30, 32, 38, 39, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68,76, 77, 78, 79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107,109, 112, 116, 125, 126, 127, 130, 132, 135, 138, 139, 143, 144, 145,147, 149, 152, 156, 157, 159, 160, 161, 162, 164, 166, 167, 168, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 181 and 182 correspondingto the positions of SEQ ID NO: 1, wherein the variant has an amino acidsequence which is at least 60%, at least 70%, at least 80%, at least 90%or at least 95% identical to SEQ ID NO: 1, wherein the variant has DNaseactivity and wherein the variant has at least one improved propertycompared to the DNase of SEQ ID NO 1.

In one aspect the invention relates to a variant according to any of thepreceding claims comprising an alteration compared to the polypeptideshown in SEQ ID NO: 1 at one or more positions corresponding topositions 1, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 19, 21, 22, 24,25, 27, 28, 29, 30, 32, 38, 39, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59,61, 63, 65, 68, 76, 77, 78, 79, 80, 82, 83, 92, 93, 94, 99, 101, 102,104, 105, 107, 109, 112, 116, 125, 126, 127, 130, 132, 135, 138, 139,143, 144, 145, 147, 149, 152, 156, 157, 159, 160, 161, 162, 164, 166,167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 181 and 182of SEQ ID NO: 1, wherein the variant has a sequence identity to theentire length of the polypeptide shown in SEQ ID NO: 1 of at least 80%and wherein the variant has DNase activity.

In some aspects, the DNase variant has improved performance wherein theimproved performance is increased stability e.g. improved detergentstability, improved in wash stability, improved storage stability and/orimproved thermo stability.

In some preferred aspects the alteration is a substitution and the DNasevariant of the invention comprises one or more substitution(s) (comparedto SEQ ID NO 1), wherein the substitution(s) is/are selected from thegroup consisting of: T1E, T1L, T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C,P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M,A10I, A10Q, A10T, A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13T,S13Q, S13F, S13R, S13V, S13N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M,Q14R, N16S, A17C, A17V, A17E, A17T, A17S, T19K, T19N, T19L, T19S, T19I,T19V, K21Q, K21E, K21M, T22P, T22A, T22V, T22D, T22R, T22K, T22M, T22E,T22H, T22L, T22W, T22F, T22C, T22S, T22I, G24Y, S25P, S25T, S27N, S27I,S27M, S27D, S27T, S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K,S30D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N,S39M, S39D, Q40V, S42G, S42C, S42D, S42L, S42M, S42F, S42N, S42W, V49R,L51I, K52I, K52Q, K52H, A55S, D56I, D56L, D56T, S57W, S57Y, S57F, S57H,S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V,S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L, T65I, T65V, T65R, T65K,S68V, S68I, S68W, S68K, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C,V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76I, V76F, T77N, T77Y,T77W, T77R, F78L, F78I, F78H, F78V, F78Y, F78C, T79G, T79R, N80K, N80S,S82L, S82E, S82K, S82R, S82H, D83C, D83F, D83L, L92T, A93G, E94N, G99S,S101D, S101A, S102M, S102L, S102V, S102A, S102K, S102T, S102R, T104S,T104P, T104A, T105V, T105I, K107L, K107C, K107R, K107H, K107S, K107M,K107E, K107A, K107Q, K107D, Q109K, Q109R, Q109S, A112S, S116D, S116R,S116Q, S116H, S116V, S116A, S116E, S116K, A125K, S126I, S126E, S126A,S126C, T127C, T127V, T127S, S130E, G132R, D135R, T138Q, W139R, R143E,R143K, S144Q, S144H, S144A, S144L, S144P, S144E, S144K, G145V, G145E,G145D, G145A, A147H, A147R, A147K, A147Q, A147W, A147N, A147S, G149S,K152H, K152R, S156C, S156G, S156K, S156R, S156T, S156A, T157S, Y159H,Y159F, K160R, K160V, W161L, W161Y, G162Q, G162N, G162D, G162M, G162R,G162A, G162S, G162E, G162L, G162K, G162V, G162H, S164R, S164H, S164N,S164T, Q166D, S167M, S167L, S167F, S167W, S167E, S167A, S167Y, S167H,S167C, S167I, S167Q, S167V, S167T, S168V, S168E, S168D, S168L, K170S,K170L, K170F, K170R, T171D, T171E, T171N, T171A, T171S, T171C, A172G,A172S, L173T, L173A, L173V, Q174L, G175D, G175E, G175N, G175R, G175S,M176H, L177I, N178D, N178E, N178T, N178S, N178A, S179E, S181R, S181E,S181D, S181I, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q, S181V,S181G, S181A, Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D,Y182Q, Y182F, Y182L, Y182N, Y182I, Y182E, Y182T and Y182W, wherein thevariant has DNase activity and wherein each position corresponds to theposition of the polypeptide shown in in SEQ ID NO: 1 (numberingaccording to SEQ ID NO 1).

In an even more preferred aspects the alteration is a substitution andthe DNase variant of the invention comprises one or more substitution(s)(compared to SEQ ID NO 1), wherein the substitution(s) is/are selectedfrom the group consisting of: T1I, T1V, T1Y, T1M, T1E, G4N, T5F, T5C,P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M,A10I, A10Q, A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13Q, S13F,S13R, S13V, S13N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S,A17C, A17V, A17E, A17T, T19K, T19L, T19S, T19I, T19V, K21E, K21M, T22P,T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,T22I, G24Y, S25P, S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L,S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P,S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42C, S42L, S42M, S42F, S42W,V49R, L51I, K52I, K52H, A55S, D56I, D56L, D56T, S57W, S57F, S57H, S57C,S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K,S59R, S59M, S59I, S59H, N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V,S68I, S68W, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H,V76E, V76A, V76Y, V76N, V76M, V76R, V76F, T77N, T77Y, T77W, T77R, F78I,F78H, F78Y, F78C, T79G, T79R, N80K, S82L, S82E, S82K, S82R, S82H, D83C,D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L, S102V,S102A, S102K, S102T, S102R, T104P, T104A, T105V, T105I, K107L, K107C,K107R, K107H, K107S, K107M, K107E, K107A, K107D, Q109R, Q109S, A112S,S116D, S116R, S116Q, S116H, S116V, S116A, S116E, S116K, A125K, S126I,S126E, S126A, S126C, T127C, T127V, S130E, G132R, D135R, T138Q, W139R,R143E, R143K, S144Q, S144H, S144L, S144P, S144E, S144K, G145V, G145E,G145D, G145A, A147Q, A147W, A147S, G149S, K152H, K152R, S156C, S156G,S156K, S156R, S156T, S156A, T157S, Y159F, K160V, W161L, W161Y, G162Q,G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K, G162V, G162H,S164R, S164T, Q166D, S167M, S167L, S167F, S167W, S167E, S167A, S167Y,S167H, S167C, S167I, S167Q, S167V, S167T, S168V, S168E, S168D, S168L,K170S, K170L, K170F, K170R, T171D, T171E, T171A, T171C, A172G, A172S,L173T, L173A, L173V, Q174L, G175D, G175E, G175N, G175R, M176H, L177I,N178D, N178E, N178T, N178S, N178A, S179E, S181R, S181E, S181D, S181F,S181H, S181W, S181L, S181M, S181Y, S181Q, S181G, S181A, Y182M, Y182C,Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N,Y182I, Y182E, Y182T and Y182W, wherein the variant has DNase activityand wherein each position corresponds to the position of the polypeptideshown in in SEQ ID NO: 1 (numbering according to SEQ ID NO 1).

In some aspects, the number of substitutions in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 substitutions.

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K,S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L,A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N,S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E,A17T, A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P,T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F,S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V,I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C,S42D, S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S,D56I, D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T,Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H,N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y,S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y,V76N, V76M, V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H,F78V, F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H,D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L,S102V, S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V, T105I,K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D,Q109K, Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A,S116E, S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S,S130E, G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A,S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H, A147R,A147K, A147Q, A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G,S156K, S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L,W161Y, G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L,G162K, G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M, S167L,S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q, S167V,S167T, S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D,T171E, T171N, T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V,Q174L, G175D, G175E, G175N, G175R, G175S, M176H, L177I, N178D, N178E,N178T, N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H,S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C,Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N,Y182I, Y182E, Y182T and Y182W, wherein the variant has DNase activity,wherein the variant has at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100% sequence identity to the polypeptide shown in in SEQ ID NO: 1and wherein each position corresponds to the position of the polypeptideshown in in SEQ ID NO: 1 (numbering according to SEQ ID NO 1).

In some aspects, the present invention relates to a DNase variant havingat least 60% sequence identity to SEQ ID NO: 1 wherein when the varianthas at least one improved property compared to a DNase comprising theamino acid sequence shown in in SEQ ID NO: 1 when tested in a relevantassay. Some aspects of the invention relate to DNase variants having animprovement factor above 1 when the DNase variants are tested for aproperty of interest in a relevant assay, wherein the property of thereference DNase is given a value of 1. In some aspects, the property isstability, such as storage stability in detergent in another aspect theproperty is wash performance, such as deep cleaning performance.

In some aspects, the variants according to the invention have one ormore improved property relative to the parent DNases measured as anHalf-life Improvement Factor (HIF) that is greater than 1.0, wherein theimproved property is stability such as stability in detergent.

The half-life improvement factor (HIF) for the variants may becalculated as T_(1/2variant)/T_(1/2reference). Improved variants wereidentified as variants having a half-life improvement factor HIF largerthan 1.0 compared to the reference sequence.

In some aspects, the variants according to the invention have anHalf-life Improvement Factor (HIF) which is at least 1.1; 1.2; 1.3; 1.4;1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7, 2.8;2.9; 3.0, 3.1; 3.2; 3.3; 3.4; 3.5, 3.6, 3.7, 3.8, 3.9; 4.0, 4.1; 4.2;4.3; 4.4; 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1; 5.2; 5.3; 5.4; 5.5, 5.6,5.7, 5.8, 5.9; 3.0, 6.1; 6.2; 6.3; 6.4; 6.5, 6.6, 6.7, 6.8, 6.9; 7.0,7.1; 7.2; 7.3; 7.4; 7.5, 7.6, 7.7, 7.8, 7.9; 8.0, 8.1; 8.2; 8.3; 8.4;8.5, 8.6, 8.7, 8.8, 8.9; 9.0, 9.1; 9.2; 9.3; 9.4; 9.5, 9.6, 9.7, 9.8,9.9; 10.0, 10.1; 10.2; 10.3; 10.4; 10.5, 10.6, 10.7, 10.8, 10.9; 12, 15,16, 20, 25 or 30.

Amino acid positions within a protein that are useful for makingvariants are those positions wherein at least one alterations lead to avariant exhibiting an improved characteristic as compared to theunchanged protein i.e. parent i.e. HIF>1.0. The improved characteristicmay be determined using the assay I or II or as described in example 2.

In some aspects, the DNase variants of the invention have improved DNaseactivity compared to a reference DNase e.g. a DNase comprising thepolypeptide shown in in SEQ ID NO: 1, wherein the activity is tested inAssay I or Assay II described in Assays and Examples below.

The activity improvement factor (AIF) is calculated as the activity ofthe variants (slope at reference conditions) divided by the activity ofthe reference. Variants with an improved AIF>1.0 was identified ashaving improved activity compared to the corresponding reference.

In some aspects, the DNase variants of the invention have improvedstability in detergent compared to a reference DNase e.g. a DNasecomprising the polypeptide shown in in SEQ ID NO: 1, wherein stabilityis tested as described in example 2.

In some aspects, the DNase variants have improved deep cleaningperformance compared to a reference DNase, e.g. a DNase comprising thepolypeptide shown in in SEQ ID NO: 1.

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K,S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L,A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N,S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E,A17T, A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P,T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F,S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V,I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C,S42D, S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S,D56I, D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T,Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H,N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y,S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y,V76N, V76M, V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H,F78V, F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H,D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L,S102V, S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V, T105I,K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D,Q109K, Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A,S116E, S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S,S130E, G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A,S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H, A147R,A147K, A147Q, A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G,S156K, S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L,W161Y, G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L,G162K, G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M, S167L,S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q, S167V,S167T, S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D,T171E, T171N, T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V,Q174L, G175D, G175E, G175N, G175R, G175S, M176H, L177I, N178D, N178E,N178T, N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H,S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C,Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N,Y182I, Y182E, Y182T and Y182W, wherein the variant has at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, such as at least 96%, at least 97%, at least98%, or at least 99%, but less than 100%, sequence identity to thepolypeptide shown in in SEQ ID NO: 1, wherein each position correspondsto the position of the polypeptide shown in in SEQ ID NO: 1 (numberingaccording to SEQ ID NO 1) and wherein the variant has at least onimproved property compared to the parent DNase e.g. a DNase comprisingthe polypeptide having the amino acid sequence shown in in SEQ ID NO 1,optionally the improved property is one or more of the following;improved DNase activity, wherein the activity is tested in Assay I orAssay II, improved stability, wherein stability is tested as describedin example 2 and/or improved deep cleaning performance.

In some aspects, the DNase variant of the invention has an improvedstability, measured as Half-life Improvement Factor, HIF, compared tothe parent or compared to the DNase having the polypeptide shown in SEQID NO: 1.

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K,S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L,A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N,S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E,A17T, A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P,T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F,S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V,I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C,S42D, S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S,D56I, D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T,Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H,N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y,S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y,V76N, V76M, V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H,F78V, F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H,D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L,S102V, S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V, T105I,K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D,Q109K, Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A,S116E, S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S,S130E, G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A,S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H, A147R,A147K, A147Q, A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G,S156K, S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L,W161Y, G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L,G162K, G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M, S167L,S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q, S167V,S167T, S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D,T171E, T171N, T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V,Q174L, G175D, G175E, G175N, G175R, G175S, M176H, L177I, N178D, N178E,N178T, N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H,S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C,Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N,Y182I, Y182E, Y182T and Y182W, wherein each substitution provides aDNase variant having an increase in stability measured as half-lifeimprovement factor, HIF, of at least 1.05, such as 1.08, such as 1.1,such as 1.15, such as 1.2, such as 1.25, such as 1.3, such as 1.4, suchas 1.5, such as 1.6, such as 1.7, such as 1.8, such as 1.9, such as 2,such as 3, such as 4, such as 5 or such as at least 10 compared to theparent DNase e.g. a DNase comprising the polypeptide having the aminoacid sequence shown in in SEQ ID NO 1, wherein the variant has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, such as at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide shown in in SEQ ID NO: 1, wherein each position correspondsto the position of the polypeptide shown in in SEQ ID NO: 1 (numberingaccording to SEQ ID NO 1).

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, T1F, T1Y, T1M, T1E, G4N, P6V, S7D, K8V, S9K, S9Q, S9V, A10D, A10M,A10I, Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N,S13H, Q14M, A17C, A17V, A17E, A17T, A17S, T19K, T19N, T19L, T19S, K21Q,K21E, T22P, T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W,T22F, T22C, T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T,S27V, S27F, S27A, S30K, S30D, S30H, S30T, D32Q, I38V, S39A, S39P, S39Y,S39H, S39E, Q40V, S42G, S42C, S42D, S42L, S42M, S42F, S42N, V49R, L51I,K52I, K52Q, A55S, D56I, D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P,S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R,S59M, S59I, P63A, T65L, T65I, T65V, S68V, S68I, S68W, S68K, S68Y, S68H,S68C, S68T, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M,V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V, T79G,T79R, N80K, N80S, S82L, S82E, S82K, S82R, D83C, D83F, L92T, A93G, G99S,S101D, S102M, S102L, S102V, S102A, S102K, T104S, T104P, T105V, T105I,K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D,Q109K, Q109R, A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E,A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E, G132R,W139R, R143E, R143K, S144Q, S144H, S144A, S144L, S144P, S144E, G145V,G145E, A147H, A147R, A147K, A147Q, A147W, A147N, A147S, G149S, K152H,K152R, S156C, S156G, S156K, S156R, T157S, Y159H, K160R, W161L, G162Q,G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K, S164R,S164H, S164N, Q166D, S167M, S167L, S167F, S167W, S167E, S167A, S167Y,S167H, S167C, S167I, S167Q, S168V, S168E, S168D, S168L, K170S, K170L,T171D, T171E, T171N, T171A, T171S, T171C, A172G, L173T, L173A, L173V,Q174L, G175D, G175E, G175N, G175R, G175S, L177I, N178D, N178E, N178T,N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H, S181W,S181L, S181M, S181Y, S181Q, S181V, S181G, Y182M, Y182C, Y182K, Y182G,Y182A, Y182S, Y182V, Y182D and Y182Q, wherein each substitution providesa DNase variant having an increase in stability measured as half-lifeimprovement factor, HIF, of at least 1.1, wherein the variant has atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, such as at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide shown in in SEQ ID NO: 1, wherein each positioncorresponds to the position of the polypeptide shown in in SEQ ID NO: 1(numbering according to SEQ ID NO 1).

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, T1F, P6V, S7D, K8V, S9K, A10D, Q12S, Q12V, S13D, S13Y, S13T, S13Q,S13F, S13R, Q14M, A17C, A17V, A17E, A17T, T19K, T19N, T19L, K21Q, K21E,T22P, T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F,T22C, T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S30K, S30D, D32Q,I38V, S39A, S39P, S39Y, Q40V, S42G, S42C, S42D, S42L, V49R, K52I, K52Q,A55S, D56I, D56L, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, Y58A,Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, P63A, T65L, S68V, S68I,S68W, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R,T77N, T77Y, T77W, T77R, F78L, F78I, T79G, T79R, N80K, S82L, S82E, D83C,A93G, S101D, S102M, T105V, T105I, K107L, K107C, K107R, K107H, K107S,K107M, K107E, K107A, Q109K, Q109R, A112S, S116D, S116R, S116Q, A125K,S126I, S126E, T127C, T127V, T127S, S130E, G132R, W139R, R143E, R143K,S144Q, S144H, S144A, S144L, S144P, G145V, G145E, A147H, A147R, A147K,A147Q, A147W, A147N, A147S, G149S, K152H, S156C, T157S, Y159H, W161L,G162Q, G162N, G162D, G162M, S164R, S164H, Q166D, S167M, S167L, S167F,S167W, S167E, S167A, S167Y, S167H, S168V, S168E, S168D, K170S, K170L,T171D, T171E, T171N, A172G, L173T, L173A, Q174L, G175D, G175E, G175N,L177I, N178D, N178E, N178T, N178S, N178A, S181R, S181E, S181D, S181I,S181F, Y182M, Y182C, Y182K, Y182G and Y182A, wherein each substitutionprovides a DNase variant having an increase in stability measured ashalf-life improvement factor, HIF, of at least 1.15, wherein the varianthas at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, such as at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide shown in in SEQ ID NO: 1, wherein eachposition corresponds to the position of the polypeptide shown in in SEQID NO: 1 (numbering according to SEQ ID NO 1).

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, T1F, P6V, K8V, A10D, Q12S, Q12V, S13D, S13Y, S13T, S13Q, A17C,A17V, T19K, K21Q, K21E, T22P, T22A, T22V, T22D, T22R, T22K, T22M, T22E,T22H, T22L, T22W, T22F, T22C, T22S, T22I, G24Y, S25P, S25T, S27N, S30K,D32Q, I38V, S39A, S39P, S39Y, Q40V, S42G, S42C, K52I, K52Q, A55S, D56I,S57W, S57Y, S57F, S57H, S57C, S57P, S57V, Y58A, S59C, S59T, S59L, S59Q,S59V, S59K, P63A, T65L, S68V, S68I, S68W, V76G, V76L, V76C, V76K, V76H,V76E, V76A, V76Y, V76N, T77N, T77Y, F78L, F78I, T79G, T79R, N80K, A93G,S101D, S102M, T105V, T105I, K107L, K107C, K107R, K107H, Q109K, Q109R,A112S, S116D, S116R, S126I, S126E, T127C, T127V, T127S, S130E, G132R,R143E, R143K, S144Q, S144H, S144A, G145V, A147H, A147R, A147K, A147Q,A147W, A147N, K152H, S156C, G162Q, G162N, G162D, S164R, Q166D, S167M,S167L, S167F, S167W, S167E, S167A, S168V, S168E, S168D, K170S, K170L,T171D, T171E, A172G, L173T, L173A, Q174L, G175D, G175E, G175N, L177I,N178D, N178E, N178T, S181R, S181E, Y182M, Y182C and Y182K, wherein eachsubstitution provides a DNase variant having an increase in stabilitymeasured as half-life improvement factor, HIF, of at least 1.2, whereinthe variant has at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, such as at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide shown in in SEQ ID NO: 1, whereineach position corresponds to the position of the polypeptide shown in inSEQ ID NO: 1 (numbering according to SEQ ID NO 1).

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,T1V, Q12S, Q12V, A17C, T22P, T22A, T22V, T22D, Q40V, K52I, K52Q, A55S,D56I, S57W, S57Y, S57F, S57H, S57C, S57P, S68V, V76G, V76L, V76C, T77N,F78L, A93G, T105V, K107L, Q109K, A112S, S126I, G132R, G145V, A147H,A147R, A147K, A147Q, S156C, G162Q, Q166D, S167M, S168V, K170S, T171D,L173T, L173A, G175D, G175E and L177I, wherein each substitution providesa DNase variant having an increase in stability measured as half-lifeimprovement factor, HIF, of at least 1.5, wherein the variant has atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, such as at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide shown in in SEQ ID NO: 1, wherein each positioncorresponds to the position of the polypeptide shown in in SEQ ID NO: 1(numbering according to SEQ ID NO 1).

In some preferred aspects, a DNase variant of the invention comprise oneor more substitution(s) (compared to SEQ ID NO 1) wherein thesubstitution(s) is/are selected from the group consisting of: T1I, T1L,Q12S, Q12V, A17C, T22P, Q40V, K52I, S57W, S57Y, S57F, V76G, Q109K,A112S, A147H, A147R, A147K, K170S, T171D and G175D, wherein eachsubstitution provides a DNase variant having an increase in stabilitymeasured as half-life improvement factor, HIF, of at least 2.0, whereinthe variant has at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, such as at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide shown in in SEQ ID NO: 1, whereineach position corresponds to the position of the polypeptide shown in inSEQ ID NO: 1 (numbering according to SEQ ID NO 1).

In some particular preferred aspects, a DNase variant of the inventioncomprise one or more substitution(s) (compared to SEQ ID NO 1) whereinthe substitution(s) is/are selected from the group consisting of: T1I,T1L, T1V, T22P, S25P, S57Y, S57W, S57F, S59V, S68V, V76L, T77Y, F78L,A93G, Q109R, S116D, T127V, S144P, A147H, A147R, G162Q, Q166D, S167L,S167F, G175D, G175N and N178D, wherein each substitution provides aDNase variant with at least one improved property compared to thepolypeptide shown in SEQ ID NO 1, wherein the variant has at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, such as at least 96%, at least 97%, at least98%, or at least 99%, but less than 100% sequence identity to thepolypeptide shown in in SEQ ID NO: 1 and wherein each positioncorresponds to the position of the polypeptide shown in in SEQ ID NO: 1(numbering according to SEQ ID NO 1).

In an embodiment, the variant has improved (increased) specific activitycompared to the parent enzyme.

In an embodiment, the variant has improved (increased) stability understorage conditions compared to the parent enzyme.

In an embodiment, the variant has improved (increased) thermostabilitycompared to the parent enzyme.

The DNase variants of the invention have at least one improved propertycompared to the parent DNase, such as a DNase having the amino acidsequence shown in SEQ ID NO 1. Each of these mutations provides at leastone improved property, such as improved stability, when introduced inSEQ ID NO 1 or polypeptides having at least 60% sequence identityhereto. It will be clear to the skilled artisan that each of thesemutations provides an improved effect thus 2, 3, 4 etc. of the abovesubstitutions may increase this effect. Thus some aspects of theinvention relate to DNase variants comprising at least two, three, four,five, six, seven, eight of the substitutions selected from the groupconsisting of selected from the group consisting of T1I, T1L, T1V, T1F,T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V,S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L, A10K,Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H,S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T,A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P, T22A,T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22S,T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A,S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M,S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C, S42D,S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S, D56I,D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A,Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D,P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y, S68H,S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N,V76M, V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V,F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H, D83C,D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L, S102V,S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V, T105I, K107L,K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D, Q109K,Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E,S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E,G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A, S144L,S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H, A147R, A147K,A147Q, A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G, S156K,S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L, W161Y,G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K,G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M, S167L, S167F,S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q, S167V, S167T,S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D, T171E,T171N, T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V, Q174L,G175D, G175E, G175N, G175R, G175S, M176H, L177I, N178D, N178E, N178T,N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H, S181W,S181L, S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C, Y182K,Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I,Y182E, Y182T and Y182W (numbering according to SEQ ID NO 1).

Preferred substitutions includes one or more of the following mutations;T1I, T1V, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K,S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10V, A10L, A10K,Q12S, Q12V, Q12E, S13D, S13Y, S13Q, S13F, S13R, S13V, S13N, S13H, S13M,S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, T19K,T19L, T19S, T19I, T19V, K21E, K21M, T22P, T22A, T22V, T22D, T22R, T22K,T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22I, G24Y, S25P, S27N, S27I,S27M, S27D, S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D,S30H, S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M,S39D, Q40V, S42C, S42L, S42M, S42F, S42W, V49R, L51I, K52I, K52H, A55S,D56I, D56L, D56T, S57W, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A,Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D,P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68Y, S68H, S68C,S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M,V76R, V76F, T77N, T77Y, T77W, T77R, F78I, F78H, F78Y, F78C, T79G, T79R,N80K, S82L, S82E, S82K, S82R, S82H, D83C, D83F, D83L, L92T, A93G, E94N,G99S, S101D, S101A, S102M, S102L, S102V, S102A, S102K, S102T, S102R,T104P, T104A, T105V, T105I, K107L, K107C, K107R, K107H, K107S, K107M,K107E, K107A, K107D, Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H,S116V, S116A, S116E, S116K, A125K, S126I, S126E, S126A, S126C, T127C,T127V, S130E, G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H,S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147Q, A147W,A147S, G149S, K152H, K152R, S156C, S156G, S156K, S156R, S156T, S156A,T157S, Y159F, K160V, W161L, W161Y, G162Q, G162D, G162M, G162R, G162A,G162S, G162E, G162L, G162K, G162V, G162H, S164R, S164T, Q166D, S167M,S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q,S167V, S167T, S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R,T171D, T171E, T171A, T171C, A172G, A172S, L173T, L173A, L173V, Q174L,G175D, G175E, G175N, G175R, M176H, L177I, N178D, N178E, N178T, N178S,N178A, S179E, S181R, S181E, S181D, S181F, S181H, S181W, S181L, S181M,S181Y, S181Q, S181G, S181A, Y182M, Y182C, Y182K, Y182G, Y182A, Y182S,Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I, Y182E or Y182T.

Particular preferred substitutions also include one or more of thefollowing; T1I, T1V, Q12S, Q12V, A17C, T22P, T22A, T22V, T22D, Q40V,K52I, A55S, D56I, S57W, S57F, S57H, S57C, S57P, S68V, V76G, V76L, V76C,T77N, A93G, T105V, K107L, A112S, S126I, G132R, G145V, A147Q, S156C,G162Q, Q166D, S167M, S168V, K170S, T171D, L173T, L173A, G175D, G175E orL177I.

Particular preferred substitutions also include one or more of thefollowing; T1I, T1V, T22P, S25P, S57W, S57F, S59V, S68V, V76L, T77Y,A93G, Q109R, S116D, T127V, S144P, G162Q, Q166D, S167L, S167F, G175D,G175N or N178D.

In some aspects, the DNase variants of the invention comprising any ofthe above mutations are effective in deep cleaning.

The variants according to the invention may comprise additionalmutations to the ones listed above. These additional modificationsshould preferably not significantly change the improved properties ofthe variant DNase.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for DNase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64.

The variants of the invention preferably comprise the conservativemotifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO:26), which are shared among DNases of the GYS-clade as described below.As explained in “Definitions” a clade comprises a group of polypeptidesclustered together based on homologous features traced to a commonancestor. Polypeptides forming a group e.g. a clade as shown in aphylogenetic tree often share common properties and are more closelyrelated than other polypeptides not in the clade.

Thus, one preferred aspect of the invention relates to a variant of aDNase parent, wherein the variant comprises one or both motifs[D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26),wherein the variant comprises one or more substitution(s) compared toSEQ ID NO 1, wherein the substitution is selected from the groupconsisting of: T1I, T1V, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D,S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q,A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13Q, S13F, S13R, S13V,S13N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V,A17E, A17T, T19K, T19L, T19S, T19I, T19V, K21E, K21M, T22P, T22A, T22V,T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22I, G24Y,S25P, S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L, S27E, G28L,Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H,S39E, S39N, S39M, S39D, Q40V, S42C, S42L, S42M, S42F, S42W, V49R, L51I,K52I, K52H, A55S, D56I, D56L, D56T, S57W, S57F, S57H, S57C, S57P, S57V,S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M,S59I, S59H, N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W,S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A,V76Y, V76N, V76M, V76R, V76F, T77N, T77Y, T77W, T77R, F78I, F78H, F78Y,F78C, T79G, T79R, N80K, S82L, S82E, S82K, S82R, S82H, D83C, D83F, D83L,L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L, S102V, S102A, S102K,S102T, S102R, T104P, T104A, T105V, T105I, K107L, K107C, K107R, K107H,K107S, K107M, K107E, K107A, K107D, Q109R, Q109S, A112S, S116D, S116R,S116Q, S116H, S116V, S116A, S116E, S116K, A125K, S126I, S126E, S126A,S126C, T127C, T127V, S130E, G132R, D135R, T138Q, W139R, R143E, R143K,S144Q, S144H, S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A,A147Q, A147W, A147S, G149S, K152H, K152R, S156C, S156G, S156K, S156R,S156T, S156A, T157S, Y159F, K160V, W161L, W161Y, G162Q, G162D, G162M,G162R, G162A, G162S, G162E, G162L, G162K, G162V, G162H, S164R, S164T,Q166D, S167M, S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C,S167I, S167Q, S167V, S167T, S168V, S168E, S168D, S168L, K170S, K170L,K170F, K170R, T171D, T171E, T171A, T171C, A172G, A172S, L173T, L173A,L173V, Q174L, G175D, G175E, G175N, G175R, M176H, L177I, N178D, N178E,N178T, N178S, N178A, S179E, S181R, S181E, S181D, S181F, S181H, S181W,S181L, S181M, S181Y, S181Q, S181G, S181A, Y182M, Y182C, Y182K, Y182G,Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I, Y182E,Y182T and Y182W, wherein the variant has a sequence identity to thepolypeptide shown in SEQ ID NO: 1 of at least 80% and the variant hasDNase activity.

The invention relates to a variant of a DNase parent, wherein thevariant comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO:25) or ASXNRSKG (SEQ ID NO: 26), wherein the variant comprises one ormore substitution(s) compared to SEQ ID NO 1, wherein the substitutionis selected from the group consisting of: T1I, T1L, T1V, T1F, T1Y, T1M,T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F,S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L, A10K, Q12S, Q12V,Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H, S13M, S13W,S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, A17S, T19K,T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P, T22A, T22V, T22D,T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22S, T22I, G24Y,S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A, S27C, S27L,S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P,S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C, S42D, S42L, S42M,S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S, D56I, D56L, D56T,S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C,S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L,T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y, S68H, S68C, S68T,S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R,V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V, F78Y, F78C,T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H, D83C, D83F, D83L,L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L, S102V, S102A, S102K,S102T, S102R, T104S, T104P, T104A, T105V, T105I, K107L, K107C, K107R,K107H, K107S, K107M, K107E, K107A, K107Q, K107D, Q109K, Q109R, Q109S,A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E, S116K, A125K,S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E, G132R, D135R,T138Q, W139R, R143E, R143K, S144Q, S144H, S144A, S144L, S144P, S144E,S144K, G145V, G145E, G145D, G145A, A147H, A147R, A147K, A147Q, A147W,A147N, A147S, G149S, K152H, K152R, S156C, S156G, S156K, S156R, S156T,S156A, T157S, Y159H, Y159F, K160R, K160V, W161L, W161Y, G162Q, G162N,G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K, G162V, G162H,S164R, S164H, S164N, S164T, Q166D, S167M, S167L, S167F, S167W, S167E,S167A, S167Y, S167H, S167C, S167I, S167Q, S167V, S167T, S168V, S168E,S168D, S168L, K170S, K170L, K170F, K170R, T171D, T171E, T171N, T171A,T171S, T171C, A172G, A172S, L173T, L173A, L173V, Q174L, G175D, G175E,G175N, G175R, G175S, M176H, L177I, N178D, N178E, N178T, N178S, N178A,S179E, S181R, S181E, S181D, S181I, S181F, S181H, S181W, S181L, S181M,S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C, Y182K, Y182G, Y182A,Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I, Y182E, Y182T andY182W (numbering according to SEQ ID NO 1) wherein the variant has asequence identity to the polypeptide shown in SEQ ID NO: 1 of at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% but less than 100% and the variant has DNase activity.

Parent DNase

Preferably the DNase parent is selected from any of the enzyme classesE.C. 3.1.11, E.C. 3.1.12, E.C. 3.1.15, E.C. 3.1.16, E.C. 3.1.21, E.03.1.22, E.0 3.1.23, E.0 3.1.24 and E.C.3.1.25.

Preferably, the DNase parent is obtained from a microorganism and theDNase is a microbial enzyme. The DNase is preferably of fungal orbacterial origin.

The DNase parent is preferably obtainable from Bacillus e.g. Bacillus,such as a Bacillus cibi, Bacillus sp-62451, Bacillus horikoshii,Bacillus sp-16840, Bacillus sp-62668, Bacillus sp-13395, Bacillushorneckiae, Bacillus sp-11238, Bacillus idriensis, Bacillus sp-62520,Bacillus algicola, Bacillus vietnamensis, Bacillus hwajinpoensis,Bacillus indicus, Bacillus marisflavi, Bacillus luciferensis, Bacillussp. SA2-6.

The DNase parent preferably belong to the group of DNases comprised inthe GYS-clade, which are DNases comprising the conservative motifs[D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26) andwhich share similar structural and functional properties, see e.galignment in FIG. 1 and generation of phylogenetic trees in example 11of WO 2017/060475. The DNases of the GYS-clade are preferably obtainedfrom Bacillus genus.

One embodiment of the invention relates to a variant of a DNase parentof the GYS-clade having DNase activity, optionally wherein the parentcomprises one or both of the motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO:25), ASXNRSKG (SEQ ID NO: 26) and wherein the polypeptide is selectedfrom the group of polypeptides:

-   -   a) a polypeptide having at least 80%, at least 85%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99% or 100% sequence identity to the polypeptide shown in SEQ ID        NO: 1,    -   b) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 2,    -   c) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 3,    -   d) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 4,    -   e) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 5,    -   f) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 6,    -   g) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 7,    -   h) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 8,    -   i) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 9,    -   j) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 10,    -   k) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 11,    -   l) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 12,    -   m) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 13,    -   n) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 14,    -   o) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 15,    -   p) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 16,    -   q) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 17,    -   r) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 18,    -   s) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 19,    -   t) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 20,    -   u) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 21,    -   v) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 22,    -   w) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 23, and    -   x) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 24.

Polypeptides having DNase activity and which comprise the GYS-clademotifs have shown particularly good deep cleaning properties e.g. theDNases are particularly effective in removing or reducing components oforganic matter, such as biofilm components, from an item such as atextile or a hard surface.

In some preferred aspect, the invention relates to a variant of a DNaseparent, wherein the DNase parent belong to the GYS-clade and wherein theparent DNase comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ IDNO: 25) or ASXNRSKG (SEQ ID NO: 26).

The parent DNases may be (a) a polypeptide having at least 60% sequenceidentity to the mature polypeptide of SEQ ID NO: 1, (b) a polypeptideencoded by a polynucleotide that hybridizes under high stringencyconditions with the mature polypeptide coding sequence or thefull-length complement hereof; or (c) a polypeptide encoded by apolynucleotide having at least 60% sequence identity to the maturepolypeptide coding sequence.

In some aspects, the parent has a sequence identity to the polypeptideshown in in SEQ ID NO: 1 of at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, suchas at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to the polypeptide shown in in SEQ ID NO: 1, whichhave DNase activity. In one aspect, the amino acid sequence of theparent differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, from the polypeptide shown in in SEQ ID NO: 1.

In some aspects, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 1.

In some aspects, the parent is a Bacillus DNase, e.g. a bacillus cibiDNase, e.g., the DNase disclosed herein as SEQ ID NO: 1.

Use DNase Variants

The DNase variants of the invention are suitable for use in cleaningsuch as laundry. Thus, some aspect of the invention relates a method forlaundering an item, wherein the method comprises the steps of:

-   -   a. Exposing an item to a wash liquor comprising a DNase variant        of the invention;    -   b. Completing at least one wash cycle; and    -   c. Optionally rinsing the item,        -   wherein the item is a textile.

The pH of the liquid solution is in the range of 1 to 11, such as in therange 5.5 to 11, such as in the range of 7 to 9, in the range of 7 to 8or in the range of 7 to 8.5.

The wash liquor may have a temperature in the range of 5° C. to 95° C.,or in the range of 10° C. to 80° C., in the range of 10° C. to 70° C.,in the range of 10° C. to 60° C., in the range of 10° C. to 50° C., inthe range of 15° C. to 40° C. or in the range of 20° C. to 30° C. Insome aspects, the temperature of the wash liquor is 30° C.

The invention further relates to an item washed according to the method.The DNase variants of the invention may be added to a wash liquor.

The concentration of the DNase variant enzyme in the wash liquor istypically in the range of 0.00004-100 ppm enzyme protein, such as in therange of 0.00008-100, in the range of 0.0001-100, in the range of0.0002-100, in the range of 0.0004-100, in the range of 0.0008-100, inthe range of 0.001-100 ppm enzyme protein, 0.01-100 ppm enzyme protein,preferably 0.05-50 ppm enzyme protein, more preferably 0.1-50 ppm enzymeprotein, more preferably 0.1-30 ppm enzyme protein, more preferably0.5-20 ppm enzyme protein, and most preferably 0.5-10 ppm enzymeprotein.

In some aspects, the DNase variants of the present invention areeffective in preventing and/or reducing the malodor. The presence ofbiofilm or EPS implies that the laundry items become sticky andtherefore soil adheres to the sticky areas. When dirty laundry items arewashed together with less dirty laundry items the dirt present in thewash liquor tend to adhere to the laundry (e.g. by re-deposition) inparticular if the laundry is sticky as described above. As a result,hereof the laundry item is more “soiled” after wash than before wash. Insome aspects, the DNase variants of the invention have improved deepcleaning properties compared to the parent DNase and in some aspects,the DNase variants of the invention reduces stickiness and/orre-deposition.

In some aspects, the invention relates to the use of a DNase variantaccording to the invention for deep cleaning of an item, wherein theitem is a fabric or a hard surface.

Further, the invention relates to the use of a DNase variant accordingto the invention for preventing and/or reducing the adherence of soil toan item. In some aspect, the item is textile. When the soil does notadhere to the item, the item appears cleaner. Thus, the inventionfurther relates to the use of a DNase variant according to the inventionfor maintaining or improving the whiteness of the item.

The present invention further relates to detergent compositionscomprising a DNase variant according to the invention preferably with adetergent adjunct ingredient. The detergent composition comprising aDNase variants according to the invention may be used for deep cleaningof an item, for preventing and/or reducing the stickiness of an item,for pretreating stains on the item, for preventing and/or reducingredeposition of soil during a wash cycle, for preventing and/or reducingadherence of soil to an item, for maintaining or improving the whitenessof an item and/or for preventing and/or reducing malodor from an item.

Preparation of Variants

The present invention also relates to a method for obtaining a DNasevariant having at least one improved property compared to the parentDNase e.g. compared to the polypeptide shown in SEQ ID NO: 1.

One aspect of the invention relates to method for obtaining a DNasevariant, comprising;

-   -   a) introducing into a parent DNase an alteration at one or more        positions corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10,        12, 13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38,        39, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76,        77, 78, 79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107,        109, 112, 116, 125, 126, 127, 130, 132, 135, 138, 139, 143, 144,        145, 147, 149, 152, 156, 157, 159, 160, 161, 162, 164, 166, 167,        168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 181 and        182 of SEQ ID NO 1, wherein the variant has DNase activity,    -   b) and recovering the variant.        The method preferably comprises introduction of 1-20, e.g. 1-10        and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations        compared to the polypeptide shown in SEQ ID NO: 1.

Some aspect of the invention relates to a method for obtaining a DNasevariant having at least one improved property compared to the parentDNase e.g. compared to the polypeptide shown in SEQ ID NO: 1, comprising

-   -   a) introducing into a parent DNase having at least 60% identity        to the polypeptide shown in SEQ ID NO: 1 a substitution at one        or more positions selected from the group consisting of: 1, 4,        5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 19, 21, 22, 24, 25, 27,        28, 29, 30, 32, 38, 39, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59,        61, 63, 65, 68, 76, 77, 78, 79, 80, 82, 83, 92, 93, 94, 99, 101,        102, 104, 105, 107, 109, 112, 116, 125, 126, 127, 130, 13, 135,        138, 139, 143, 144, 145, 147, 149, 152, 156, 157, 159, 160, 161,        162, 164, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177,        178, 179, 181 and 182, wherein the variant has at least 60%, at        least 70%, at least 80%, at least 85%, at least 90% or at least        95% sequence identity to the polypeptide shown in SEQ ID NO: 1;        and    -   b) recovering the variant.

The present invention further relates to a method for obtaining a DNasevariant comprising introducing at least one alteration compared to SEQID NO: 1, wherein the alteration is a substitution and wherein thesubstitution is selected from the group consisting of: T1I, T1L, T1V,T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q,S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L, A10K,Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H,S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T,A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P, T22A,T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22S,T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A,S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M,S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C, S42D,S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S, D56I,D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A,Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D,P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y, S68H,S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N,V76M, V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V,F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H, D83C,D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L, S102V,S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V, T105I, K107L,K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D, Q109K,Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E,S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E,G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A, S144L,S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H, A147R, A147K,A147Q, A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G, S156K,S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L, W161Y,G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K,G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M, S167L, S167F,S167W, S167E, S167A, S167Y, S167H, S167C, S167I, S167Q, S167V, S167T,S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D, T171E,T171N, T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V, Q174L,G175D, G175E, G175N, G175R, G175S, M176H, L177I, N178D, N178E, N178T,N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H, S181W,S181L, S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C, Y182K,Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I,Y182E, Y182T and Y182W compared to the polypeptide shown in SEQ ID NO:1.

Some aspects of the invention relate to a method for obtaining a DNasevariant, comprising introducing into a parent DNase an alteration at oneor more positions, wherein the DNase parent belong to the GYS-clade andwherein the parent DNase comprises one or both of the motifs[D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26).

Some aspects of the invention relate to a method for obtaining a DNasevariant, comprising introducing into a parent DNase an alteration at oneor more positions, wherein the parent DNase is selected from the groupof polypeptides:

-   -   a) a polypeptide having at least 80%, at least 85%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99% or 100% sequence identity to the polypeptide shown in SEQ ID        NO: 1,    -   b) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 2,    -   c) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 3,    -   d) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 4,    -   e) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 5,    -   f) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 6,    -   g) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 7,    -   h) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 8,    -   i) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 9,    -   j) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 10,    -   k) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 11,    -   l) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 12,    -   m) a polypeptide having at least 80%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the polypeptide        shown in SEQ ID NO: 13,    -   n) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 14,    -   o) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 15,    -   p) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 16,    -   q) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 17,    -   r) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 18,    -   s) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 19,    -   t) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 20,    -   u) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 21,    -   v) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 22,    -   w) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 23, and    -   x) a polypeptide having at least 80% at least 80%, at least 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99% or 100% sequence identity to the polypeptide shown in        SEQ ID NO: 24.

Some aspect relates to a method of obtaining a DNase variant wherein theparent DNase is obtained from bacillus genus.

Some aspect relates to a method of obtaining a DNase variant wherein theDNase parent has at least 80%, such as at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99% butless than 100% sequence identity to the polypeptide shown in SEQ ID NO:1.

The variants of the invention may be prepared by procedures such asthose mentioned below.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding the DNase variants of the present inventionoperably linked to one or more control sequences that direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Bacillus nidulansacetamidase, Bacillus niger neutral alpha-amylase, Bacillus niger acidstable alpha-amylase, Bacillus niger or Bacillus awamori glucoamylase(glaA), Bacillus cibi TAKA amylase, Bacillus cibi alkaline protease,Bacillus cibi triose phosphate isomerase, Fusarium oxysporumtrypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase(WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusariumvenenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucormiehei aspartic proteinase, Trichoderma reesei beta-glucosidase,Trichoderma reesei cellobiohydrolase I, Trichoderma reeseicellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichodermareesei endoglucanase II, Trichoderma reesei endoglucanase III,Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2 tpi promoter (a modified promoterfrom an Bacillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Bacillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Bacillus niger neutral alpha-amylase gene in which theuntranslated leader has been replaced by an untranslated leader from anBacillus nidulans or Bacillus cibi triose phosphate isomerase gene); andmutant, truncated, and hybrid promoters thereof. Other promoters aredescribed in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO 1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3 phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3phosphoglycerate kinase. Other useful promoters for yeast host cells aredescribed by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′ terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Bacillus nidulans acetamidase, Bacillus nidulansanthranilate synthase, Bacillus niger glucoamylase, Bacillus nigeralpha-glucosidase, Bacillus cibi TAKA amylase, Fusarium oxysporumtrypsin-like protease, Trichoderma reesei beta-glucosidase, Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseV, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, andTrichoderma reesei translation elongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3phosphate dehydrogenase. Other useful terminators for yeast host cellsare described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene. Examples of suitable mRNA stabilizer regions areobtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and aBacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′ terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Bacillus cibi TAKA amylase and Bacillus nidulans triosephosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO 1), Saccharomyces cerevisiae 3phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Bacillus nidulans anthranilate synthase,Bacillus niger glucoamylase, Bacillus niger alpha-glucosidase Bacilluscibi TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′ end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′ end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Bacillus niger neutral amylase, Bacillus niger glucoamylase,Bacillus cibi TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N terminus of apolypeptide and the signal peptide sequence is positioned next to the Nterminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Bacillus niger glucoamylase promoter, Bacilluscibi TAKA alpha-amylase promoter, and Bacillus cibi glucoamylasepromoter, Trichoderma reesei cellobiohydrolase I promoter, andTrichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding the DNase variants of the presentinvention, a promoter, and transcriptional and translational stopsignals. The various nucleotide and control sequences may be joinedtogether to produce a recombinant expression vector that may include oneor more convenient restriction sites to allow for insertion orsubstitution of the polynucleotide encoding the polypeptide at suchsites. Alternatively, the polynucleotide may be expressed by insertingthe polynucleotide or a nucleic acid construct comprising thepolynucleotide into an appropriate vector for expression. In creatingthe expression vector, the coding sequence is located in the vector sothat the coding sequence is operably linked with the appropriate controlsequences for expression. In a further aspect, polynucleotide sequencecodons have been modified by nucleotide substitutions to correspond tothe codon usage of the host organism intended for production of thepolypeptide of the present invention. The recombinant expression vectormay be any vector (e.g., a plasmid or virus) that can be convenientlysubjected to recombinant DNA procedures and can bring about expressionof the polynucleotide. The choice of the vector will typically depend onthe compatibility of the vector with the host cell into which the vectoris to be introduced. The vector may be a linear or closed circularplasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′ phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in a Bacillus cell are Bacillusnidulans or Bacillus cibi amdS and pyrG genes and a Streptomyceshygroscopicus bar gene. Preferred for use in a Trichoderma cell areadeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In some aspects, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome. For integration into thehost cell genome, the vector may rely on the polynucleotide's sequenceencoding the polypeptide or any other element of the vector forintegration into the genome by homologous or non-homologousrecombination. Alternatively, the vector may contain additionalpolynucleotides for directing integration by homologous recombinationinto the genome of the host cell at a precise location(s) in thechromosome(s). To increase the likelihood of integration at a preciselocation, the integrational elements should contain a sufficient numberof nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 basepairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Bacillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Bacillusawamori, Bacillus foetidus, Bacillus fumigatus, Bacillus japonicus,Bacillus nidulans, Bacillus niger, Bacillus cibi, Bjerkandera adusta,Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsisgilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa,Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus,Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Bacillus and Trichoderma host cells are described inEP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a DNasevariant of the present invention, comprising (a) cultivating a cell,under conditions conducive for production of the DNase variants; andoptionally, (b) recovering the DNase variant. In some aspects, the cellis a Bacillus cell. In another aspect, the cell is a Bacillus cibi cell.

The present invention also relates to methods of producing a DNasevariant of the present invention, comprising (a) cultivating arecombinant host cell of the present invention under conditionsconducive for production of the DNase variant; and optionally, (b)recovering the DNase variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The DNase variant may be detected using methods known in the art thatare specific for the DNase variant polypeptide. These detection methodsinclude, but are not limited to, use of specific antibodies, formationof an enzyme product, or disappearance of an enzyme substrate. Forexample, an enzyme assay may be used to determine the activity of thepolypeptide.

The DNase variant polypeptide may be recovered using methods known inthe art. For example, the DNase variant polypeptide may be recoveredfrom the nutrient medium by conventional procedures including, but notlimited to, collection, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation. In some aspects, afermentation broth comprising the DNase variant is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the variant.

Compositions

The present invention further relates to a detergent compositioncomprising at least one DNase variant according to the invention andpreferably a detergent adjunct ingredient. The detergent composition maybe used for improving deep-cleaning effect, including but not limited todeep cleaning of an item, preventing and/or reducing the stickiness ofan item, for pretreating stains on the item, for preventing and/orreducing redeposition of soil during a wash cycle, for preventing and/orreducing adherence of soil to an item, for maintaining or improving thewhiteness of an item and for preventing and/or reducing malodor from anitem. The DNase variants of the invention are useful in powder andliquid detergent.

Some aspects of the invention relates to a detergent compositioncomprising a DNase variant which compared to the polypeptide shown inSEQ ID NO: 1 comprises an alteration at one or more positions selectedfrom the group consisting of: 1, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16,17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38, 39, 40, 42, 49, 51, 52,55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78, 79, 80, 82, 83, 92, 93,94, 99, 101, 102, 104, 105, 107, 109, 112, 116, 125, 126, 127, 130, 13,135, 138, 139, 143, 144, 145, 147, 149, 152, 156, 157, 159, 160, 161,162, 164, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 181 and 182 and a detergent adjunct.

In some aspects of the invention, the detergent composition comprises aDNase variant which compared to the polypeptide shown in SEQ ID NO: 1comprises one or more substitutions selected from the group consistingof: T1I, T1L, T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D,S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q,A10T, A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F,S13R, S13V, S13N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R, N16S,A17C, A17V, A17E, A17T, A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q,K21E, K21M, T22P, T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L,T22W, T22F, T22C, T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D,S27T, S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H,S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D,Q40V, S42G, S42C, S42D, S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I,K52Q, K52H, A55S, D56I, D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P,S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R,S59M, S59I, S59H, N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I,S68W, S68K, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H,V76E, V76A, V76Y, V76N, V76M, V76R, V76I, V76F, T77N, T77Y, T77W, T77R,F78L, F78I, F78H, F78V, F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E,S82K, S82R, S82H, D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D,S101A, S102M, S102L, S102V, S102A, S102K, S102T, S102R, T104S, T104P,T104A, T105V, T105I, K107L, K107C, K107R, K107H, K107S, K107M, K107E,K107A, K107Q, K107D, Q109K, Q109R, Q109S, A112S, S116D, S116R, S116Q,S116H, S116V, S116A, S116E, S116K, A125K, S126I, S126E, S126A, S126C,T127C, T127V, T127S, S130E, G132R, D135R, T138Q, W139R, R143E, R143K,S144Q, S144H, S144A, S144L, S144P, S144E, S144K, G145V, G145E, G145D,G145A, A147H, A147R, A147K, A147Q, A147W, A147N, A147S, G149S, K152H,K152R, S156C, S156G, S156K, S156R, S156T, S156A, T157S, Y159H, Y159F,K160R, K160V, W161L, W161Y, G162Q, G162N, G162D, G162M, G162R, G162A,G162S, G162E, G162L, G162K, G162V, G162H, S164R, S164H, S164N, S164T,Q166D, S167M, S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C,S167I, S167Q, S167V, S167T, S168V, S168E, S168D, S168L, K170S, K170L,K170F, K170R, T171D, T171E, T171N, T171A, T171S, T171C, A172G, A172S,L173T, L173A, L173V, Q174L, G175D, G175E, G175N, G175R, G175S, M176H,L177I, N178D, N178E, N178T, N178S, N178A, S179E, S181R, S181E, S181D,S181I, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q, S181V, S181G,S181A, Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q,Y182F, Y182L, Y182N, Y182I, Y182E, Y182T and Y182W and a detergentadjunct.

In some aspects of the invention, the detergent adjunct ingredient isselected from the group consisting of surfactants, builders,flocculating aid, chelating agents, dye transfer inhibitors, enzymes,enzyme stabilizers, enzyme inhibitors, catalytic materials, bleachactivators, hydrogen peroxide, sources of hydrogen peroxide, preformedperacids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, structure elasticizing agents, fabric softeners, carriers,hydrotropes, builders and co-builders, fabric hueing agents,anti-foaming agents, dispersants, processing aids, and/or pigments.

The detergent adjunct ingredient may be a surfactant. One advantage ofincluding a surfactant in a detergent composition comprising a DNasevariant is that the wash performance is improved. In some aspects, thedetergent adjunct ingredient is a builder or a clay soilremoval/anti-redeposition agent.

In some aspects, detergent adjunct ingredient is an enzyme. Thedetergent composition may comprise one or more enzymes, as specifiedbelow. The one or more enzymes may be selected from the group consistingof proteases, amylases, lipases, cutinases, cellulases, endoglucanases,xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes,haloperoxygenases, catalases and mannanases. Specific enzymes suitablefor the detergent compositions of the invention are described below.

The detergent composition may be formulated as a bar, a homogenoustablet, and a tablet having two or more layers, a pouch having one ormore compartments, a regular or compact powder, a granule, a paste, agel, or a regular, compact or concentrated liquid. The detergentcomposition can be a liquid detergent, a powder detergent or a granuledetergent.

Some aspects of the present invention relate to laundry or cleaningcompositions comprising a DNase, preferably at a level of from about0.000001 wt % to about 1 wt %, from about 0.0001 wt % to about 1 wt %,from about 0.0002 wt % to about 1 wt %, from about 0.0005 wt % to about1 wt %, from about 0.001 wt % to about 1 wt %, from about 0.002 wt % toabout 1 wt %, from about 0.005 wt % to about 1 wt %, preferably fromabout 0.01 wt % to about 0.5 wt %, preferably from 0.0002 wt % to about1 wt % by weight (wt %) of the composition. The amounts are wt % perunit active enzyme e.g. from about 0.00001 wt % to about 1 wt % of DNaseby weight of the composition.

The concentration of the active enzyme having DNase activity ispreferably at least 0.00001%, preferably at least 0.00002%, preferablyat least 0.0001 wt %, preferably at least 0.0002 wt %, preferably atleast 0.001 wt %, preferably at least 0.002 wt %, preferably at least0.005 wt %, preferably at least 0.01 wt %, preferably at least 0.02 wt%, preferably at least 0.05 wt % preferably at least 0.1 wt % of thetotal detergent concentration.

The amount enzyme may also be in ppm (mg/L) active enzyme protein. Thus,in one aspect 30 the amount of DNase in the composition is at least0.00001 ppm, 0.00002 ppm, 0.00005 ppm, 0.0001 ppm, 0.0002 ppm, 0.0005ppm, 0.001 ppm, 0.002 ppm, 0.005 ppm, 0.01 ppm, 0.02 ppm, 0.05 ppm, 0.1ppm, 0.2 ppm, 0.5 ppm, 1 ppm, 2 ppm, 5 ppm, 10 ppm or at least 20 ppmDNase enzymes. In one aspect, the amount of DNase in the composition isin the range from about 0.00001 ppm to about 10 ppm, or in the rangefrom about 0.0001 ppm to about 2 ppm or in the range from about 0.001ppm to 35 about 2 ppm DNase enzymes.

In some aspects, the detergent composition is a liquid or powder laundrydetergent, suitable for e.g. washing at high temperature and/or pH, suchas at or above 40° C. and/or at or above pH 8. In some aspects, thedetergent composition is a liquid or powder laundry detergent, suitablefor e.g. washing at low temperature and/or pH, such as at or below 20°C. and/or pH 6. The detergent may also be formulated as a unit dosedetergent and/or compact detergent optionally with minimum or no water.The detergent may also be a dish wash detergent. The laundry and dishwash detergents may be phosphate-free.

Liquid Detergent Composition

The DNase variants of the invention may also be formulated in liquidlaundry compositions such as a liquid laundry compositions compositioncomprising:

-   -   a) at least 0.00001 e.g. 0.005 mg of active DNase variant        protein per Liter detergent, and optionally    -   b) 0 wt % to 60 wt % of at least one surfactant, and optionally    -   c) 0 wt % to 50 wt % of at least one builder

The surfactant may be selected among nonionic, anionic and/or amphotericsurfactants as described above, preferably anionic or nonionicsurfactants but also amphoteric surfactants may be used. In general,bleach-stable surfactants are preferred. Preferred anionic surfactantsare sulphate surfactants and in particular alkyl ether sulphates,especially C-9-15 alcohol ethersulfates, C12-15 primary alcoholethoxylate, C8-C16 ester sulphates and C10-C14 ester sulphates, such asmono dodecyl ester sulphates Non-limiting examples of anionicsurfactants include sulfates and sulfonates, in particular, linearalkylbenzenesulfonates (LAS), isomers of LAS, branchedalkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or salt offatty acids (soap), and combinations thereof.

The anionic surfactants are preferably added to the detergent in theform of salts. Suitable cations in these salts are alkali metal ions,such as sodium, potassium and lithium and ammonium salts, for example(2-hydroxyethyl) ammonium, bis(2-hydroxyethyl) ammonium andtris(2-hydroxyethyl) ammonium salts. Non-limiting examples of nonionicsurfactants include alcohol ethoxylates (AE or AEO), alcoholpropoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acidalkyl esters, such as ethoxylated and/or propoxylated fatty acid alkylesters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE),alkylpolyglycosides (APG), alkoxylated amines, fatty acidmonoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylatedfatty acid monoethanolamides (EFAM), propoxylated fatty acidmonoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acylN-alkyl derivatives of glucosamine (glucamides, GA, or fatty acidglucamides, FAGA), as well as products available under the trade namesSPAN and TWEEN, and combinations thereof. Commercially availablenonionic surfactants includes Plurafac™, Lutensol™ and Pluronic™ fromBASF, Dehypon™ series from Cognis and Genapol™ series from Clariant.

The builder is preferably selected among phosphates, sodium citratebuilders, sodium carbonate, sodium silicate, sodium aluminosilicate(zeolite). Suitable builders are alkali metal or ammonium phosphates,polyphosphates, phosphonates, polyphosphates, carbonates, bicarbonates,borates, citrates, and polycarboxylates. Citrate builders, e.g., citricacid and soluble salts thereof (particularly sodium salt), arepolycarboxylate builders. Citrates can be used in combination withzeolite, silicates like the BRITESIL types, and/or layered silicatebuilders. The builder is preferably added in an amount of about 0-65% byweight, such as about 5% to about 50% by weight. In a laundry detergent,the level of builder is typically about 40-65% by weight, particularlyabout 50-65% by weight, particularly from 20% to 50% by weight. Thebuilder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in cleaning detergents may beutilized. Non-limiting examples of builders include zeolites,diphosphates (pyrophosphates), triphosphates such as sodium triphosphate(STP or STPP), carbonates such as sodium carbonate, soluble silicatessuch as sodium metasilicate, layered silicates (e.g., SKS-6 fromHoechst), and (carboxymethyl)inulin (CMI), and combinations thereof.Further non-limiting examples of builders include citrate, chelatorssuch as aminocarboxylates, aminopolycarboxylates and phosphonates, andalkyl- or alkenylsuccinic acid. Additional specific examples include2,2′,2″-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid(IDS), ethylenediamine-N,N′-disuccinic acid (EDDS),methylglycine-N,N-diacetic acid (MGDA), glutamic acid-N,N-diacetic acid(GLDA), 1-hydroxyethane-1,1-diphosphonic acid,N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(sulfomethylglutamic acid (SMGL), N-(2-sulfoethyl)-glutamicacid (SEGL), N-methyliminodiacetic acid (MI DA), serine-N,N-diaceticacid (SEDA), isoserine-N,N-diacetic acid (ISDA),phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diaceticacid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA),taurine-N,N-diacetic acid (TUDA) andN″-(2-hydroxyethypethylenediamine-N,N,N′-triacetic acid (HEDTA),diethanolglycine (DEG), and combinations and salts thereof. Phosphonatessuitable for use herein include 1-hydroxyethane-1,1-diphosphonic acid(HEDP), ethylenediaminetetrakis (methylenephosphonicacid) (EDTMPA),diethylenetriaminepentakis (methylenephosphonic acid) (DTMPA or DTPMPAor DTPMP), nitrilotris (methylenephosphonic acid) (ATMP or NTMP),2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC),hexamethylenediaminetetrakis (methylenephosphonic acid) (HDTMP).

The composition may also contain 0-50% by weight, such as about 5% toabout 30%, of a detergent co-builder. The detergent composition mayinclude a co-builder alone, or in combination with a builder, forexample a zeolite builder. Non-limiting examples of co-builders includehomopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly (acrylic acid/maleic acid) (PAA/PMA) orpolyaspartic acid. Further exemplary builders and/or co-builders aredescribed in, e.g., WO 09/102854, U.S. Pat. No. 5,977,053

In some aspects, the builder is a non-phosphorus based builder such ascitric acid and/or methylglycine-N,N-diacetic acid (MGDA) and/orglutamic-N,N-diacetic acid (GLDA) and/or salts thereof.

The liquid composition may also be phosphate free in that instance thepreferred builders includes citrate and/or methylglycine-N,N-diaceticacid (MGDA) and/or glutamic-N,N-diacetic acid (GLDA) and/or saltsthereof.

-   Some aspects of the invention relate to a liquid laundry composition    comprising:    -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per Liter of        composition, and optionally    -   b) 0% to 30% by weight of at least one surfactant wherein the at        least one surfactant is selected from LAS, AEOS and/or SLES, and        optionally    -   c) 0% to 50% by weight of at least one builder selected from        citrate and/or methylglycine-N,N-diacetic acid (MGDA) and/or        glutamic-N,N-diacetic acid (GLDA) and/or salts thereof.-   Some aspects of the invention relate to a liquid laundry composition    comprising:    -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per Liter of        composition, and optionally    -   b) 0% to 30% by weight of at least one surfactant wherein the at        least one surfactant is LAS, AEOS and/or SLES, and optionally    -   c) 0% to 50% by weight of at least one builder selected from H        EDP, DTMPA or DTPMPA.

The liquid detergent composition may typically contain at least 20% byweight and up to 95% water, such as up to 70% water, up to 50% water, upto 40% water, up to 30% water, up to 20% water, up to 10% water, up to5% water or up to 2% water. Other types of liquids, including withoutlimitation, alkanols, amines, diols, ethers and polyols may be includedin an aqueous liquid detergent. An aqueous liquid detergent may containfrom 0-30% organic solvent. A liquid detergent may even be non-aqueous,wherein the water content is below 10%, preferably below 5%.

Powder Compositions

The detergent composition may also be formulated into a granulardetergent for laundry or dish wash. Some aspects of the inventionconcern a granular detergent composition comprising

-   -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per gram of        composition, optionally    -   b) 0 wt % to 40 wt % anionic surfactants, optionally    -   c) 0 wt % to 20 wt % nonionic surfactants, and/or optionally    -   d) 0 wt % to 40 wt % builder such as carbonates, zeolites,        phosphate builder, calcium sequestering builders or complexing        agents.

The surfactant may be selected among nonionic, anionic and/or amphotericsurfactants as described above, preferably anionic or nonionicsurfactants but also amphoteric surfactants may be used. In general,bleach-stable surfactants are preferred. Preferred anionic surfactantsare sulphate surfactants and in particular alkyl ether sulphates,especially C-9-15 alcohol ethersulfates, C12-15 primary alcoholethoxylate, C8-C16 ester sulphates and C10-C14 ester sulphates, such asmono dodecyl ester sulphates Non-limiting examples of anionicsurfactants include sulfates and sulfonates, in particular, linearalkylbenzenesulfonates (LAS), isomers of LAS, branchedalkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or salt offatty acids (soap), and combinations thereof. The anionic surfactantsare preferably added to the detergent in the form of salts. Suitablecations in these salts are alkali metal ions, such as sodium, potassiumand lithium and ammonium salts, for example (2-hydroxyethyl) ammonium,bis(2-hydroxyethyl) ammonium and tris(2-hydroxyethyl) ammonium salts.

Non-limiting examples of nonionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fattyacid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides,GA, or fatty acid glucamides, FAGA), as well as products available underthe trade names SPAN and TWEEN, and combinations thereof.

Commercially available nonionic surfactants include Plurafac™, Lutensol™and Pluronic™ range from BASF, Dehypon™ series from Cognis and Genapol™series from Clariant.

The builder is may be non-phosphate such as citrate preferably as asodium salt and/or zeolites. Phosphonate builder may be any of thosedescribed above.

The builder is preferably selected among phosphates and sodium citratebuilders, sodium carbonate, sodium silicate, sodium aluminosilicate(zeolite) as described above. Suitable builders are described above andinclude alkali metal or ammonium phosphates, polyphosphates,phosphonates, polyphosphonates, carbonates, bicarbonates, borates,polyhydroxysulfonates, polyacetates, carboxylates, citrates, andpolycarboxylates. Citrate builders, e.g., citric acid and soluble saltsthereof (particularly sodium salt), are polycarboxylate builders. Thebuilder is preferably added in an amount of about 0-65% by weight, suchas about 5% to about 50% by weight, such as 5 to 40% by weight, such as10 to 40% by weight, such as 10 to 30% by weight, such as 15 to 20% byweight or such as 20 to 40% by weight. The builder may be a phosphonatebuilder including 1-hydroxyethane-1,1-diphosphonic acid (HEDP),ethylenediaminetetra (methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis (methylenephosphonic acid) (DTMPA or DTPMPA),diethylenetriamine penta (methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), 2-phosphonobutane-1,2,4-tricarboxylicacid (PBTC) and hexamethylenediaminetetra (methylenephosphonic acid)(HDTMP). Preferred phosphonates include 1-hydroxyethane-1,1-diphosphonicacid (HEDP) and/or diethylenetriaminepentakis (methylenephosphonic acid)(DTMPA or DTPMPA). The phosphonate is preferably added in an amount ofabout in a level of from about 0.01% to about 10% by weight, preferablyfrom 0.1% to about 5% by weight, more preferably from 0.5% to 3% byweight of the composition.

The granular composition may also be phosphate free in that instance thepreferred builders includes citrate and/or methylglycine-N,N-diaceticacid (MGDA) and/or glutamic-N,N-diacetic acid (GLDA) and/or saltsthereof.

-   Some aspects of the invention relate to a granular composition    comprising:    -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per gram of        composition, and optionally    -   b) 0% to 30% by weight of at least one surfactant wherein the        surfactant is LAS, AEOS and/or SLES, and optionally    -   c) 0% to 50% by weight of at least one builder selected from        citrate and/or methylglycine-N,N-diacetic acid (MGDA) and/or        glutamic-N,N-diacetic acid (GLDA) and/or salts thereof.-   Some aspects of the invention relate to a granular composition    comprising:    -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per gram of        composition, and optionally    -   b) 0% to 30% by weight of at least one surfactant wherein the at        least one surfactant is LAS, AEOS and/or SLES, and optionally    -   c) 0% to 50% by weight of at least one builder selected from H        EDP, DTM PA or DTPM PA.

The detergent may contain 0-30% by weight, such as about 1% to about20%, of a bleaching system. Any bleaching system comprising componentsknown in the art for use in cleaning detergents may be utilized.Suitable bleaching system components include sources of hydrogenperoxide; sources of peracids; and bleach catalysts or boosters.

Sources of hydrogen peroxide: Suitable sources of hydrogen peroxide areinorganic persalts, including alkali metal salts such as sodiumpercarbonate and sodium perborates (usually mono- or tetrahydrate), andhydrogen peroxide—urea (1/1).

Sources of peracids: Peracids may be (a) incorporated directly aspreformed peracids or (b) formed in situ in the wash liquor fromhydrogen peroxide and a bleach activator (perhydrolysis) or (c) formedin situ in the wash liquor from hydrogen peroxide and a perhydrolase anda suitable substrate for the latter, e.g., an ester.

a) Suitable preformed peracids include, but are not limited to,peroxycarboxylic acids such as peroxybenzoic acid and itsring-substituted derivatives, peroxy-a-naphthoic acid, peroxyphthalicacid, peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproicacid [phthalimidoperoxyhexanoic acid (PAP)], ando-carboxybenzamidoperoxycaproic acid; aliphatic and aromaticdiperoxydicarboxylic acids such as diperoxydodecanedioic acid,diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid,2-decyldiperoxybutanedioic acid, and diperoxyphthalic, -isophthalic and-terephthalic acids; perimidic acids; peroxymonosulfuric acid;peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; andmixtures of said compounds. It is understood that the peracids mentionedmay in some cases be best added as suitable salts, such as alkali metalsalts (e.g., Oxone®) or alkaline earth-metal salts.

b) Suitable bleach activators include those belonging to the class ofesters, amides, imides, nitriles or anhydrides and, where applicable,salts thereof. Suitable examples are tetraacetylethylenediamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),sodium 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), sodium4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoic acid (DOBA),sodium 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO98/17767. A particular family of bleach activators of interest wasdisclosed in EP624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that they are environmentally friendly.Furthermore, acetyl triethyl citrate and triacetin have goodhydrolytical stability in the product upon storage and are efficientbleach activators. Finally, ATC is multifunctional, as the citratereleased in the perhydrolysis reaction may function as a builder.

Bleach catalysts and boosters: The bleaching system may also include ableach catalyst or booster. Some non-limiting examples of bleachcatalysts that may be used in the compositions of the present inventioninclude manganese oxalate, manganese acetate, manganese-collagen,cobalt-amine catalysts and manganese triazacyclononane (MnTACN)catalysts; particularly preferred are complexes of manganese with1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), in particularMe3-TACN, such as the dinuclear manganese complex[(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and[2,2′,2″-nitrilotris(ethane-1,2-diylazanylylidene-κN-methanylylidene)triphenolato-κ3O]manganese(III).The bleach catalysts may also be other metal compounds, such as iron orcobalt complexes.

In some aspects, where a source of a peracid is included, an organicbleach catalyst or bleach booster may be used having one of thefollowing formulae:

(iii) and mixtures thereof; wherein each R1 is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R1 is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R1 isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl,hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl.

Other exemplary bleaching systems are described, e.g. in WO 2007/087258,WO 2007/087244, WO 2007/087259, EP 1867708 (Vitamin K) and WO2007/087242. Suitable photobleaches may for example be sulfonated zincor aluminium phthalocyanines.

According to some aspects and any of the previous aspects the inventionalso relates to a cleaning composition comprising;

-   -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per gram of        composition, optionally    -   b) 10-50 wt % builder and optionally    -   c) at least one bleach component, wherein the bleach is        preferably a peroxide and the bleach catalyst is a manganese        compound.

The oxygen bleach is preferably percarbonate and the manganese catalystpreferably 1,4,7-trimethyl-1,4,7-triazacyclononane or manganese (III)acetate tetrahydrate

According to some aspects and any of the previous aspects the inventionalso relates to a cleaning composition comprising;

-   -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per gram of        composition, optionally    -   b) 10-50 wt % builder selected from citric acid, methyl        glycine-N,N-diacetic acid (MGDA) and/or glutamic-N,N-diacetic        acid (GLDA) and mixtures thereof, and optionally    -   c) at least one bleach component, wherein the bleach is an        oxygen bleach and the bleach catalyst is a manganese compound.

The oxygen bleach is preferably percarbonate and the manganese catalystpreferably 1,4,7-trimethyl-1,4,7-triazacyclo-nonane or manganese (II)acetate tetrahydrate.

According to some aspects and any of the previous aspects the inventionalso relates to a detergent composition comprising;

-   -   a) at least 0.00001 e.g. 0.005 mg of DNase variant per gram of        composition, optionally    -   b) 10-50 wt % builder selected from citric acid, methyl        glycine-N,N-diacetic acid (MGDA) and/or glutamic-N,N-diacetic        acid (GLDA) and mixtures thereof, and optionally    -   c) 0.1-40 wt %, preferably from 0.5-30 wt %, of bleaching        components, wherein the bleach components are a peroxide,        preferably percabonate and a metal-containing bleach catalyst        preferably 1,4,7-trimethyl-1,4,7-triazacyclononane or        manganese (II) acetate tetrahydrate (MnTACN).

The choice of detergent components may include, for textile care, theconsideration of the type of textile to be cleaned, the type and/ordegree of soiling, the temperature at which cleaning is to take place,and the formulation of the detergent product. Although componentsmentioned below are categorized by general header according to afunctionality, this is not to be construed as a limitation, as acomponent may comprise additional functionalities as will be appreciatedby the skilled artisan, including the exemplary non-limiting componentsshown in below.

Hydrotropes

The detergent composition may contain 0-10% by weight, for example 0-5%by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of ahydrotrope. Any hydrotrope known in the art for use in detergents may beutilized. Non-limiting examples of hydrotropes include sodiumbenzenesulfonate, sodium p-toluene sulfonate (STS), sodium xylenesulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate,amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate,sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, andcombinations thereof.

Polymers

The detergent composition may contain 0-10% by weight, such as 0.5-5%,2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art foruse in detergents may be utilized. The polymer may function as aco-builder as mentioned above, or may provide antiredeposition, fibreprotection, soil release, dye transfer inhibition, grease cleaningand/or anti-foaming properties. Some polymers may have more than one ofthe above-mentioned properties and/or more than one of thebelow-mentioned motifs. Exemplary polymers include(carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethyleneoxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin(CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid,and lauryl methacrylate/acrylic acid copolymers, hydrophobicallymodified CMC (HM-CMC) and silicones, copolymers of terephthalic acid andoligomeric glycols, copolymers of poly(ethylene terephthalate) andpoly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole)(PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) andpolyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymersinclude sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent composition of the present invention may also includefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions andthus altering the tint of said fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight. In contrast, fabric hueing agents alter the tint of a surface asthey absorb at least a portion of the visible light spectrum. Suitablefabric hueing agents include dyes and dye-clay conjugates, and may alsoinclude pigments. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group consisting of dyes falling into the Colour Index(C.I.) classifications of Direct Blue, Direct Red, Direct Violet, AcidBlue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO 2005/03274, WO2005/03275, WO 2005/03276 and EP 1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt%, or even from about 0.0001 wt % to about 0.04 wt % fabric hueingagent. The composition may comprise from 0.0001 wt % to 0.2 wt % fabrichueing agent, this may be especially preferred when the composition isin the form of a unit dose pouch. Suitable hueing agents are alsodisclosed in, e.g. WO 2007/087257 and WO 2007/087243.

Enzymes

The detergent composition may comprise one or more additional enzymessuch as a protease, lipase, cutinase, amylase, carbohydrase, cellulase,pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., alaccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

Cellulases

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and WO 99/001544.

Other cellulases are endo-beta-1,4-glucanase enzyme having a sequence ofat least 97% identity to the amino acid sequence of position 1 toposition 773 of SEQ ID NO: 2 of WO 2002/099091 or a family 44xyloglucanase, which a xyloglucanase enzyme having a sequence of atleast 60% identity to positions 40-559 of SEQ ID NO: 2 of WO2001/062903.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S) Carezyme Premium™ (Novozymes A/S), Celluclean™(Novozymes A/S), Celluclean Classic™ (Novozymes A/S), Cellusoft™(Novozymes A/S), Whitezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™(Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Proteases

Suitable proteases include those of bacterial, fungal, plant, viral oranimal origin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO92/19729,WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452,WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263,WO11/036264, especially the variants with substitutions in one or moreof the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74,85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128,154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198,199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255,256, 268 and 269 wherein the positions correspond to the positions ofthe Bacillus Lentus protease shown in SEQ ID NO 1 of WO 2016/001449.More preferred the protease variants may comprise one or more of thefollowing mutations: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R,S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R, G96S, G96A, S97G,S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A,V1021, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, N1205, 5126L,P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E,N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V1991, Y203W, 5206G,L211Q, L211D, N212D, N2125, M2165, A226V, K229L, Q230H, Q239R, N246K,N255W, N255D, N255E, L256E, L256D T268A or R269H. The protease variantsare preferably variants of the Bacillus Lentus protease (Savinase®)shown in SEQ ID NO 1 of WO 2016/001449 or the Bacillusamylolichenifaciens protease (BPN') shown in SEQ ID NO 2 ofWO2016/001449. The protease variants preferably have at least 80%sequence identity to the polypeptide shown in SEQ ID NO 1 or SEQ ID NO 2of WO 2016/001449.

A protease variant comprising a substitution at one or more positionscorresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO: 1 ofWO2004/067737, wherein said protease variant has a sequence identity ofat least 75% but less than 100% to SEQ ID NO: 1 of WO2004/067737.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect Ox®, Purafect OxP®,Puramax®, FN2®, FN3®, FN4®, Excellase®, Excellenz P1000™, ExcellenzP1250™, Eraser®, Preferenz P100™, Purafect Prime®, Preferenz P110™,Effectenz P1000™, Purafect®™, Effectenz P1050™, Purafect Ox®™, EffectenzP2000™, Purafast®, Properase®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases:

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP 305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamedto Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyceslipases (WO 10/065455), cutinase from Magnaporthe grisea (WO 10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO 11/084412), Geobacillus stearothermophiluslipase (WO 11/084417), lipase from Bacillus subtilis (WO 11/084599), andlipase from Streptomyces griseus (WO 11/150157) and S. pristinaespiralis(WO 12/137147).

Other examples are lipase variants such as those described in EP 407225,WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO00/34450, WO 00/60063, WO 01/92502, WO 07/87508 and WO 09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO 10/111143), acyltransferase fromMycobacterium smegmatis (WO 05/56782), perhydrolases from the CE 7family (WO 09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO 10/100028).

Amylases

Suitable amylases which can be used together with the DNases of theinvention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 1 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase obtained from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylaseobtained from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

M 197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N19S, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID NO. 2 of WO 96/023873 for numbering. More preferred variants arethose having a deletion in two positions selected from 181, 182, 183 and184, such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T16S, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T16SI, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

-   N128C+K178L+T182G+Y305R+G475K;-   N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;-   S125A+N128C+K178L+T182G+Y305R+G475K; or-   S125A+N128C+T131I+T16S I+K178L+T182G+Y305R+G475K wherein the    variants are C-terminally truncated and optionally further comprises    a substitution at position 243 and/or a deletion at position 180    and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO 2013/001078 and WO 2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes A/S), and Rapidase™ Purastar™/Effectenz™, Powerase andPreferenz S100 (from Genencor International Inc./DuPont).

Peroxidases/Oxidases

A peroxidase according to the invention is a peroxidase enzyme comprisedby the enzyme classification EC 1.11.1.7, as set out by the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (IUBMB), or any fragment obtained therefrom, exhibitingperoxidase activity.

Suitable peroxidases include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinopsis, e.g., fromC. cinerea (EP 179,486), and variants thereof as those described in WO93/24618, WO 95/10602, and WO 98/15257.

A peroxidase also includes a haloperoxidase enzyme, such aschloroperoxidase, bromoperoxidase and compounds exhibitingchloroperoxidase or bromoperoxidase activity. Haloperoxidases areclassified according to their specificity for halide ions.Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochloritefrom chloride ions.

In an aspect, the haloperoxidase is a chloroperoxidase. Preferably, thehaloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containinghaloperoxidase. In a preferred method, the vanadate-containinghaloperoxidase is combined with a source of chloride ion.

Haloperoxidases have been isolated from many different fungi, inparticular from the fungus group dematiaceous hyphomycetes, such asCaldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C.verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such asPseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S.aureofaciens.

In a preferred aspect, the haloperoxidase is derivable from Curvulariasp., in particular Curvularia verruculosa or Curvularia inaequalis, suchas C. inaequalis CBS 102.42 as described in WO 95/27046; or C.verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO97/04102; or from Drechslera hartlebii as described in WO 01/79459,Dendryphiella salina as described in WO 01/79458, Phaeotrichoconiscrotalarie as described in WO 01/79461, or Geniculosporium sp. asdescribed in WO 01/79460.

An oxidase may include any laccase enzyme comprised by the enzymeclassification EC 1.10.3.2, or any fragment obtained therefromexhibiting laccase activity, or a compound exhibiting a similaractivity, such as a catechol oxidase (EC 1.10.3.1), an o-aminophenoloxidase (EC 1.10.3.4), or a bilirubin oxidase (EC 1.3.3.5).

Preferred laccase enzymes are enzymes of microbial origin. The enzymesmay be obtained from plants, bacteria or fungi (including filamentousfungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strainof Bacillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia,Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radiata (WO 92/01046), or Coriolus, e.g., C.hirsutus (JP 2238885).

Suitable examples from bacteria include a laccase derivable from astrain of Bacillus.

A laccase obtained from Coprinopsis or Myceliophthora is preferred; alaccase obtained from Coprinopsis cinerea, as disclosed in WO 97/08325;or from Myceliophthora thermophila, as disclosed in WO 95/33836.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly (ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Other Materials

Any detergent components known in the art for use in the cleaningcomposition of the invention may also be utilized. Other optionaldetergent components include anti-corrosion agents, anti-shrink agents,anti-soil redeposition agents, anti-wrinkling agents, bactericides,binders, corrosion inhibitors, disintegrants/disintegration agents,dyes, enzyme stabilizers (including boric acid, borates, CMC, and/orpolyols such as propylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in detergents may be utilized. The choice of suchingredients is well within the skill of the artisan.

Dispersants

The cleaning compositions of the present invention can also containdispersants. In particular, powdered detergents may comprisedispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents

The cleaning compositions of the present invention may also include oneor more dye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

Fluorescent Whitening Agent

The detergent composition may preferably also contain additionalcomponents that may tint articles being cleaned, such as fluorescentwhitening agent or optical brighteners. Where present the brightener ispreferably at a level of about 0.01% to about 0.5%. Any fluorescentwhitening agent suitable for use in a laundry detergent composition maybe used in the composition of the present invention. The most commonlyused fluorescent whitening agents are those belonging to the classes ofdiaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivativesand bisphenyl-distyryl derivatives. Examples of thediaminostilbene-sulfonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulfonate, 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulfonate,4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulfonate,4,4′-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2′-disulfonate andsodium5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate.Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBSavailable from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is thedisodium salt of 4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulfonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl)-disulfonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is a4.4′-bis-(sulfostyryl)-biphenyl disodium salt also known as DisodiumDistyrylbiphenyl Disulfonate. Other fluorescers suitable for use in theinvention include the 1-3-diaryl pyrazolines and the7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % toupper levels of 0.5 or even 0.75 wt %.

Soil Release Polymers

The detergent compositions may also include one or more soil releasepolymers which aid the removal of soils from fabrics such as cotton andpolyester based fabrics, in particular the removal of hydrophobic soilsfrom polyester based fabrics. The soil release polymers may for examplebe nonionic or anionic terephthalte based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyesterpolyamides see for example Chapter 7 in Powdered Detergents, Surfactantscience series volume 71, Marcel Dekker, Inc. Another type of soilrelease polymers are amphiphilic alkoxylated grease cleaning polymerscomprising a core structure and a plurality of alkoxylate groupsattached to that core structure. The core structure may comprise apolyalkylenimine structure or a polyalkanolamine structure as describedin detail in WO 2009/087523 (hereby incorporated by reference).Furthermore random graft co-polymers are suitable soil release polymers.Suitable graft co-polymers are described in more detail in WO2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated byreference). Other soil release polymers are substituted polysaccharidestructures especially substituted cellulosic structures such as modifiedcellulose derivatives such as those described in EP 1867808 or WO2003/040279 (both are hereby incorporated by reference). Suitablecellulosic polymers include cellulose, cellulose ethers, celluloseesters, cellulose amides and mixtures thereof. Suitable cellulosicpolymers include anionically modified cellulose, nonionically modifiedcellulose, cationically modified cellulose, zwitterionically modifiedcellulose, and mixtures thereof. Suitable cellulosic polymers includemethyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxylethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methylcellulose, and mixtures thereof.

Anti-Redeposition Agents

The cleaning compositions of the present invention may also include oneor more anti-redeposition agents such as carboxymethylcellulose (CMC),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethyleneand/or polyethyleneglycol (PEG), homopolymers of acrylic acid,copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Rheology Modifiers

The cleaning compositions of the present invention may also include oneor more rheology modifiers, structurants or thickeners, as distinct fromviscosity reducing agents. The rheology modifiers are selected from thegroup consisting of non-polymeric crystalline, hydroxy-functionalmaterials, polymeric rheology modifiers which impart shear thinningcharacteristics to the aqueous liquid matrix of a liquid detergentcomposition. The rheology and viscosity of the detergent can be modifiedand adjusted by methods known in the art, for example as shown in EP2169040.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a regular orcompact powder, a granule, a paste, a gel, or a regular, compact orconcentrated liquid.

Detergent formulation forms: Layers (same or different phases), Pouches,versus forms for Machine Dosing Unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxyprpyl methyl cellulose (HPMC). Preferably the level of polymer inthe film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blend compositions comprising hydrolytically degradableand water soluble polymer blends such as polyactide and polyvinylalcohol (known under the Trade reference M8630 as sold by Chris CraftIn. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethyleneglycerol, Propylene glycol, sorbitol and mixtures thereof. The pouchescan comprise a solid laundry cleaning composition or part componentsand/or a liquid cleaning composition or part components separated by thewater soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids. Ref: (US2009/0011970 A1)

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

-   Definition/Characteristics of the Forms:

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

A liquid or gel detergent may be non-aqueous.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO 09/092699, EP1705241, EP 1382668, WO 07/001262, U.S. Pat. No. 6,472,364, WO 04/074419or WO 09/102854. Other useful detergent formulations are described in WO09/124162, WO 09/124163, WO 09/117340, WO 09/117341, WO 09/117342, WO09/072069, WO 09/063355, WO 09/132870, WO 09/121757, WO 09/112296, WO09/112298, WO 09/103822, WO 09/087033, WO 09/050026, WO 09/047125, WO09/047126, WO 09/047127, WO 09/047128, WO 09/021784, WO 09/010375, WO09/000605, WO 09/122125, WO 09/095645, WO 09/040544, WO 09/040545, WO09/024780, WO 09/004295, WO 09/004294, WO 09/121725, WO 09/115391, WO09/115392, WO 09/074398, WO 09/074403, WO 09/068501, WO 09/065770, WO09/021813, WO 09/030632, and WO 09/015951.

WO 2011025615, WO 2011016958, WO 2011005803, WO 2011005623, WO2011005730, WO 2011005844, WO 2011005904, WO 2011005630, WO 2011005830,WO 2011005912, WO 2011005905, WO 2011005910, WO 2011005813, WO2010135238, WO 2010120863, WO 2010108002, WO 2010111365, WO 2010108000,WO 2010107635, WO 2010090915, WO 2010033976, WO 2010033746, WO2010033747, WO 2010033897, WO 2010033979, WO 2010030540, WO 2010030541,WO 2010030539, WO 2010024467, WO 2010024469, WO 2010024470, WO2010025161, WO 2010014395, WO 2010044905,

WO 2010145887, WO 2010142503, WO 2010122051, WO 2010102861, WO2010099997, WO 2010084039, WO 2010076292, WO 2010069742, WO 2010069718,WO 2010069957, WO 2010057784, WO 2010054986, WO 2010018043, WO2010003783, WO 2010003792,

WO 2011023716, WO 2010142539, WO 2010118959, WO 2010115813, WO2010105942, WO 2010105961, WO 2010105962, WO 2010094356, WO 2010084203,WO 2010078979, WO 2010072456, WO 2010069905, WO 2010076165, WO2010072603, WO 2010066486, WO 2010066631, WO 2010066632, WO 2010063689,WO 2010060821, WO 2010049187, WO 2010031607, WO 2010000636.

Formulation of Enzyme in Co-Granule

The DNase may be formulated as a granule for example as a co-granulethat combines one or more enzymes. Each enzyme will then be present inmore granules securing a more uniform distribution of enzymes in thedetergent. This also reduces the physical segregation of differentenzymes due to different particle sizes. Methods for producingmulti-enzyme co-granulate for the detergent industry is disclosed in theIP.com disclosure IPCOM000200739D.

Another example of formulation of enzymes using co-granulates aredisclosed in WO 2013/188331, which relates to a detergent compositioncomprising (a) a multi-enzyme co-granule; (b) less than 10 wt zeolite(anhydrous basis); and (c) less than 10 wt phosphate salt (anhydrousbasis), wherein said enzyme co-granule comprises from 10 to 98 wt %moisture sink components and the composition additionally comprises from20 to 80 wt % detergent moisture sink components. WO 2013/188331 alsorelates to a method of treating and/or cleaning a surface, preferably afabric surface comprising the steps of (i) contacting said surface withthe detergent composition as claimed and described herein in aqueouswash liquor, (ii) rinsing and/or drying the surface.

-   The invention is further described in the following non-limiting    paragraphs.-   Paragraph 1. A DNase variant, comprising a substitution at one or    more positions corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10,    12, 13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38, 39,    40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78,    79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112,    116, 125, 126, 127, 130, 132, 135, 138, 139, 143, 144, 145, 147,    149, 152, 156, 157, 159, 160, 161, 162, 164, 166, 167, 168, 170,    171, 172, 173, 174, 175, 176, 177, 178, 179, 181 and 182 of the    polypeptide of SEQ ID NO: 1, wherein the variant has DNase activity    and wherein the variant has at least at least 60%, at least 65%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 95%, at least 96%, at least 97%, at least 98%, or at least    99%, but less than 100% sequence identity to the polypeptide shown    in SEQ ID NO: 1.-   Paragraph 2. The variant according to paragraph 1 wherein the    variant has increased stability measured as half-life improvement    factor, HIF, compared to the polypeptide shown in SEQ ID NO: 1.-   Paragraph 3. The variant according to any of the proceeding claims    wherein the variant has an improvement factor of at least 1.05, of    at least 1.10, of at least 1.15, of at least 1.20, of at least 1.50,    of at least 2.00, of at least 2.50, of at least 3.00, of at least    4.00, of at least 5.00, of at least 6.00, of at least 7.00, of at    least 8.00, of at least 9.00 or of at least 10.00.-   Paragraph 4. The variant according to any of the previous    paragraphs, comprising one or more substitution(s) selected from the    group consisting of: T1I, T1L, T1V, T1F, T1Y, T1M, T1E, G4N, T5F,    T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R,    A10D, A10M, A10I, A10Q, A10T, A10V, A10L, A10K, Q12S, Q12V, Q12E,    S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H, S13M, S13W,    S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T, A17S,    T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P, T22A,    T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,    T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V,    S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H, S30T,    D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M, S39D,    Q40V, S42G, S42C, S42D, S42L, S42M, S42F, S42N, S42W, V49R, L51I,    K52I, K52Q, K52H, A55S, D56I, D56L, D56T, S57W, S57Y, S57F, S57H,    S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q,    S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L, T65I, T65V,    T65R, T65K, S68V, S68I, S68W, S68K, S68Y, S68H, S68C, S68T, S68L,    V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R,    V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V, F78Y,    F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H, D83C,    D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L,    S102V, S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V,    T105I, K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A,    K107Q, K107D, Q109K, Q109R, Q109S, A112S, S116D, S116R, S116Q,    S116H, S116V, S116A, S116E, S116K, A125K, S126I, S126E, S126A,    S126C, T127C, T127V, T127S, S130E, G132R, D135R, T138Q, W139R,    R143E, R143K, S144Q, S144H, S144A, S144L, S144P, S144E, S144K,    G145V, G145E, G145D, G145A, A147H, A147R, A147K, A147Q, A147W,    A147N, A147S, G149S, K152H, K152R, S156C, S156G, S156K, S156R,    S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L, W161Y,    G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L,    G162K, G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M,    S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S167I,    S167Q, S167V, S167T, S168V, S168E, S168D, S168L, K170S, K170L,    K170F, K170R, T171D, T171E, T171N, T171A, T171S, T171C, A172G,    A172S, L173T, L173A, L173V, Q174L, G175D, G175E, G175N, G175R,    G175S, M176H, L177I, N178D, N178E, N178T, N178S, N178A, S179E,    S181R, S181E, S181D, S181I, S181F, S181H, S181W, S181L, S181M,    S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C, Y182K, Y182G,    Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y182I,    Y182E, Y182T and Y182W, wherein the positions correspond to amino    acid positions in the amino acid sequence shown in SEQ ID NO: 1    (numbering according to SEQ ID NO 1); and wherein the variant has    DNase activity and wherein the variant has at least at least 60%, at    least 65%, at least 70%, at least 75%, at least 80%, at least 85%,    at least 90%, at least 95%, at least 96%, at least 97%, at least    98%, or at least 99%, but less than 100% sequence identity to the    polypeptide shown in SEQ ID NO: 1.-   Paragraph 5. The variant according to any of the previous    paragraphs, comprising one or more substitution(s) selected from the    group consisting of: T1I, T1L, T1V, T1F, T1Y, T1M, T1E, G4N, P6V,    S7D, K8V, S9K, S9Q, S9V, A10D, A10M, A10I, Q12S, Q12V, Q12E, S13D,    S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H, Q14M, A17C, A17V,    A17E, A17T, A17S, T19K, T19N, T19L, T19S, K21Q, K21E, T22P, T22A,    T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,    T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T, S27V,    S27F, S27A, S30K, S30D, S30H, S30T, D32Q, I38V, S39A, S39P, S39Y,    S39H, S39E, Q40V, S42G, S42C, S42D, S42L, S42M, S42F, S42N, V49R,    L51I, K52I, K52Q, A55S, D56I, D56L, D56T, S57W, S57Y, S57F, S57H,    S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L, S59Q,    S59V, S59K, S59R, S59M, S59I, P63A, T65L, T65I, T65V, S68V, S68I,    S68W, S68K, S68Y, S68H, S68C, S68T, V76G, V76L, V76C, V76K, V76H,    V76E, V76A, V76Y, V76N, V76M, V76R, V76I, V76F, T77N, T77Y, T77W,    T77R, F78L, F78I, F78H, F78V, T79G, T79R, N80K, N80S, S82L, S82E,    S82K, S82R, D83C, D83F, L92T, A93G, G99S, S101D, S102M, S102L,    S102V, S102A, S102K, T104S, T104P, T105V, T105I, K107L, K107C,    K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D, Q109K,    Q109R, A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E,    A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E,    G132R, W139R, R143E, R143K, S144Q, S144H, S144A, S144L, S144P,    S144E, G145V, G145E, A147H, A147R, A147K, A147Q, A147W, A147N,    A147S, G149S, K152H, K152R, S156C, S156G, S156K, S156R, T157S,    Y159H, K160R, W161L, G162Q, G162N, G162D, G162M, G162R, G162A,    G162S, G162E, G162L, G162K, S164R, S164H, S164N, Q166D, S167M,    S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S167I,    S167Q, S168V, S168E, S168D, S168L, K170S, K170L, T171D, T171E,    T171N, T171A, T171S, T171C, A172G, L173T, L173A, L173V, Q174L,    G175D, G175E, G175N, G175R, G175S, L177I, N178D, N178E, N178T,    N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F, S181H,    S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, Y182M, Y182C,    Y182K, Y182G, Y182A, Y182S, Y182V, Y182D and Y182Q, wherein each    substitution at the one or more positions provides a DNase variant    having an increase in stability measured as half-life improvement    factor, HIF, of at least 1.1, wherein the variant has at least 60%,    at least 65%, at least 70%, at least 75%, at least 80%, at least    85%, at least 90%, at least 95%, such as at least 96%, at least 97%,    at least 98%, or at least 99%, but less than 100% sequence identity    to the polypeptide shown in in SEQ ID NO: 1, wherein each position    corresponds to the position of the polypeptide shown in SEQ ID NO: 1    (numbering according to SEQ ID NO 1).-   Paragraph 6. The variant according to any of the previous    paragraphs, comprising one or more substitution(s) selected from the    group consisting of: T1I, T1L, T1V, T1F, P6V, S7D, K8V, S9K, A10D,    Q12S, Q12V, S13D, S13Y, S13T, S13Q, S13F, S13R, Q14M, A17C, A17V,    A17E, A17T, T19K, T19N, T19L, K21Q, K21E, T22P, T22A, T22V, T22D,    T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C, T22S, T22I,    G24Y, S25P, S25T, S27N, S27I, S27M, S30K, S30D, D32Q, I38V, S39A,    S39P, S39Y, Q40V, S42G, S42C, S42D, S42L, V49R, K52I, K52Q, A55S,    D56I, D56L, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, Y58A,    Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, P63A, T65L, S68V,    S68I, S68W, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N,    V76M, V76R, T77N, T77Y, T77W, T77R, F78L, F78I, T79G, T79R, N80K,    S82L, S82E, D83C, A93G, S101D, S102M, T105V, T105I, K107L, K107C,    K107R, K107H, K107S, K107M, K107E, K107A, Q109K, Q109R, A112S,    S116D, S116R, S116Q, A125K, S126I, S126E, T127C, T127V, T127S,    S130E, G132R, W139R, R143E, R143K, S144Q, S144H, S144A, S144L,    S144P, G145V, G145E, A147H, A147R, A147K, A147Q, A147W, A147N,    A147S, G149S, K152H, S156C, T157S, Y159H, W161L, G162Q, G162N,    G162D, G162M, S164R, S164H, Q166D, S167M, S167L, S167F, S167W,    S167E, S167A, S167Y, S167H, S168V, S168E, S168D, K170S, K170L,    T171D, T171E, T171N, A172G, L173T, L173A, Q174L, G175D, G175E,    G175N, L177I, N178D, N178E, N178T, N178S, N178A, S181R, S181E,    S181D, S181I, S181F, Y182M, Y182C, Y182K, Y182G and Y182A, wherein    each substitution at the one or more positions provides a DNase    variant having an increase in thermo-stability measured as half-life    improvement factor, HIF, of at least 1.15, wherein the variant has    at least 60%, at least 65%, at least 70%, at least 75%, at least    80%, at least 85%, at least 90%, at least 95%, such as at least 96%,    at least 97%, at least 98%, or at least 99%, but less than 100%    sequence identity to the polypeptide shown in in SEQ ID NO: 1,    wherein each position corresponds to the position of the polypeptide    shown in SEQ ID NO: 1 (numbering according to SEQ ID NO 1).-   Paragraph 7. The variant according to any of the previous    paragraphs, comprising one or more substitution(s) selected from the    group consisting of: T1I, T1L, T1V, T1F, P6V, K8V, A10D, Q12S, Q12V,    S13D, S13Y, S13T, S13Q, A17C, A17V, T19K, K21Q, K21E, T22P, T22A,    T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F, T22C,    T22S, T22I, G24Y, S25P, S25T, S27N, S30K, D32Q, I38V, S39A, S39P,    S39Y, Q40V, S42G, S42C, K52I, K52Q, A55S, D56I, S57W, S57Y, S57F,    S57H, S57C, S57P, S57V, Y58A, S59C, S59T, S59L, S59Q, S59V, S59K,    P63A, T65L, S68V, S68I, S68W, V76G, V76L, V76C, V76K, V76H, V76E,    V76A, V76Y, V76N, T77N, T77Y, F78L, F78I, T79G, T79R, N80K, A93G,    S101D, S102M, T105V, T105I, K107L, K107C, K107R, K107H, Q109K,    Q109R, A112S, S116D, S116R, S126I, S126E, T127C, T127V, T127S,    S130E, G132R, R143E, R143K, S144Q, S144H, S144A, G145V, A147H,    A147R, A147K, A147Q, A147W, A147N, K152H, S156C, G162Q, G162N,    G162D, S164R, Q166D, S167M, S167L, S167F, S167W, S167E, S167A,    S168V, S168E, S168D, K170S, K170L, T171D, T171E, A172G, L173T,    L173A, Q174L, G175D, G175E, G175N, L177I, N178D, N178E, N178T,    S181R, S181E, Y182M, Y182C and Y182K, wherein each substitution at    the one or more positions provides a DNase variant having an    increase in thermo-stability measured as half-life improvement    factor, HIF, of at least 1.2, wherein the variant has at least 60%,    at least 65%, at least 70%, at least 75%, at least 80%, at least    85%, at least 90%, at least 95%, such as at least 96%, at least 97%,    at least 98%, or at least 99%, but less than 100% sequence identity    to the polypeptide shown in in SEQ ID NO: 1, wherein each position    corresponds to the position of the polypeptide shown in SEQ ID NO: 1    (numbering according to SEQ ID NO 1).-   Paragraph 8. The variant according to any of the previous    paragraphs, comprising one or more substitution(s) selected from the    group consisting of: T1I, T1L, T1V, Q12S, Q12V, A17C, T22P, T22A,    T22V, T22D, Q40V, K52I, K52Q, A55S, D56I, S57W, S57Y, S57F, S57H,    S57C, S57P, S68V, V76G, V76L, V76C, T77N, F78L, A93G, T105V, K107L,    Q109K, A112S, S126I, G132R, G145V, A147H, A147R, A147K, A147Q,    S156C, G162Q, Q166D, S167M, S168V, K170S, T171D, L173T, L173A,    G175D, G175E and L177I, wherein each substitution at the one or more    positions provides a DNase variant having an increase in    thermo-stability measured as half-life improvement factor, HIF, of    at least 1.5, wherein the variant has at least 60%, at least 65%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 95%, such as at least 96%, at least 97%, at least 98%, or    at least 99%, but less than 100% sequence identity to the    polypeptide shown in in SEQ ID NO: 1, wherein each position    corresponds to the position of the polypeptide shown in in SEQ ID    NO: 1 (numbering according to SEQ ID NO 1).-   Paragraph 9. The variant according to any of the previous    paragraphs, comprising one or more substitution(s) selected from the    group consisting of: T1I, T1L, Q12S, Q12V, A17C, T22P, Q40V, K52I,    S57W, S57Y, S57F, V76G, Q109K, A112S, A147H, A147R, A147K, K170S,    T171D and G175D, wherein each substitution at the one or more    positions provides a DNase variant having an increase in    thermo-stability measured as half-life improvement factor, HIF, of    at least 2.0, wherein the variant has at least 60%, at least 65%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 95%, such as at least 96%, at least 97%, at least 98%, or    at least 99%, but less than 100% sequence identity to the    polypeptide shown in in SEQ ID NO: 1, wherein each position    corresponds to the position of the polypeptide shown in in SEQ ID    NO: 1 (numbering according to SEQ ID NO 1).-   Paragraph 10. The variants of any of paragraphs 1-9, which has at    least 60%, at least 65%, at least 70%, at least 75%, at least 80%,    at least 85%, at least 90%, at least 91%, at least 92%, at least    93%, at least 94%, at least 95% identity, at least 96%, at least    97%, at least 98%, or at least 99%, but less than 100%, sequence    identity to the amino acid sequence shown in SEQ ID NO: 1.-   Paragraph 11. The variant of any of paragraphs 1-10, wherein the    number of substitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2,    3, 4, 5, 6, 7, 8, 9 or 10 substitutions.-   Paragraph 12. A polynucleotide encoding the variant of any of    paragraphs 1-11.-   Paragraph 13. A nucleic acid construct comprising the polynucleotide    of paragraph 12.-   Paragraph 14. An expression vector comprising the polynucleotide of    paragraph 12.-   Paragraph 15. A recombinant host cell comprising the polynucleotide    of paragraph 12.-   Paragraph 16. A method of producing a DNase variant, comprising:    cultivating the host cell of paragraph 15 under conditions suitable    for expression of the variant; and optionally recovering the    variant.-   Paragraph 17. A composition comprising a variant of any of the    paragraphs 1-11.-   Paragraph 18. The composition of paragraph 17, further comprising at    least one carbohydrate-active enzymes like carbohydrase, pectinase,    mannanase, amylase, cellulase, arabinase, galactanase, xylanase,    protease such as metalloproteases, lipase, a, cutinase, oxidase,    e.g., a laccase, and/or peroxidase.-   Paragraph 19 The composition according to paragraph 18, comprising a    protease wherein the protease comprises substitutions in one or more    of the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66,    74, 85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127,    128, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189,    193, 198, 199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230,    239, 246, 255, 256, 268 and 269, wherein the positions correspond to    the positions of the Bacillus Lentus protease shown in SEQ ID NO 1    of WO 2016/001449 and wherein the protease variants has at least 80%    sequence identity to the Savinase or BPN' protease shown in SEQ ID    NO 1 and SEQ ID NO 2 respectively in WO2016/001449.-   Paragraph 20 The composition according to any of paragraphs 18 or    19, comprising a protease wherein the protease comprises one or more    of the following substitution(s): S3T, V4I, S9R, S9E, A15T, S24G,    S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S,    N85R G96S, G96A, S97G, S97D, 597A, S97SD, S99E, S99D, S99G, S99M,    S99N, S99R, S99H, S101A, V1021, V102Y, V102N, S104A, G116V, G116R,    H118D, H118N, N1205, 5126L, P127Q, S128A, S154D, A156E, G157D,    G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P,    G189E, V193M, N198D, V1991, Y203W, 5206G, L211Q, L211D, N212D,    N2125, M2165, A226V, K229L, Q230H, Q239R, N246K, N255W, N255D,    N255E, L256E, L256D T268A or R269H, wherein the positions correspond    to the positions of the Bacillus lentus protease shown in SEQ ID NO    1 of WO 2016/001449 and wherein the protease variants has at least    80% sequence identity to the Savinase or BPN' protease shown in SEQ    ID NO 1 and SEQ ID NO 2 respectively in WO2016/001449.-   Paragraph 21 The composition according to any of paragraphs 18 to    20, comprising a protease wherein the protease is selected from    protease variant comprising a substitution at one or more positions    corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO:    1 of WO2004/067737, wherein said protease variant has a sequence    identity of at least 75% but less than 100% to the polypeptide shown    in SEQ ID NO: 1 of WO2004/067737.-   Paragraph 22 The composition according to any of paragraphs 18 to    21, comprising an amylase wherein the amylase is one or more of the    following amylases;    -   a) Amylases having SEQ ID NO: 2 in WO 95/10603 or variants        having 90% sequence identity to SEQ ID NO: 1 thereof. Preferred        variants are described in WO 94/02597, WO 94/18314, WO 97/43424        and SEQ ID NO: 4 of WO 99/019467, such as variants with        substitutions in one or more of the following positions: 15, 23,        105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197,        201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and        444;    -   b) amylases having SEQ ID NO: 6 in WO 02/010355 or variants        thereof having 90% sequence identity to SEQ ID NO: 6. Preferred        variants of SEQ ID NO: 6 are those having a deletion in        positions 181 and 182 and a substitution in position 193;    -   c) hybrid alpha-amylase comprising residues 1-33 of the        alpha-amylase obtained from B. amyloliquefaciens shown in SEQ ID        NO: 6 of WO 2006/066594 and residues 36-483 of the B.        licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO        2006/066594 or variants having 90% sequence identity thereof,        wherein preferred variants of this hybrid alpha-amylase are        those having a substitution, a deletion or an insertion in one        of more of the following positions: G48, T49, G107, H156, A181,        N190, M197, 1201, A209 and Q264, preferred variants of the        hybrid alpha-amylase comprising residues 1-33 of the        alpha-amylase obtained from B. amyloliquefaciens shown in SEQ ID        NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4        include those having the substitutions:-   M197T, H156Y+A181T+N190F+A209V+Q264S, or-   G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S;    -   d) amylases having SEQ ID NO: 6 shown in WO 99/019467 or        variants thereof having 90% sequence identity to SEQ ID NO: 6,        preferred variants of SEQ ID NO: 6 include those having a        substitution, a deletion or an insertion in one or more of the        following positions: R181, G182, H183, G184, N195, I206, E212,        E216 and K269, particularly preferred amylases are those having        deletion in positions R181 and G182, or positions H183 and G184;    -   e) amylases having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or        SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90%        sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or        SEQ ID NO: 7 respectively, wherein preferred variants of SEQ ID        NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those        having a substitution, a deletion or an insertion in one or more        of the following positions: 140, 181, 182, 183, 184, 195, 206,        212, 243, 260, 269, 304 and/or 476, using SEQ ID NO. 2 of WO        96/023873 for numbering, more preferred variants are those        having a deletion in two positions selected from 181, 182, 183        and 184, such as 181 and 182, 182 and 183, or positions 183 and        184, more preferred amylase variants of SEQ ID NO: 1, SEQ ID NO:        2 or SEQ ID NO: 7 are those having a deletion in positions 183        and 184 and a substitution in one or more of positions 140, 195,        206, 243, 260, 304 and/or 476;    -   f) amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10        in WO 01/66712 or variants thereof having 90% sequence identity        to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ        ID NO: 10 in WO 01/66712, preferred variants of SEQ ID NO: 10 in        WO 01/66712 include those having a substitution, a deletion or        an insertion in one of more of the following positions: 176,        177, 178, 179, 190, 201, 207, 211 and/or 264;    -   g) amylases having SEQ ID NO: 2 of WO 09/061380 or variants        having 90% sequence identity to SEQ ID NO: 2, preferred variants        of SEQ ID NO: 2 are those having a truncation of the C-terminus        and/or a substitution, a deletion or an insertion in one of more        of the following positions: Q87, Q98, S125, N128, T131, T16S,        K178, R180, S181, T182, G183, M201, F202, N225, S243, N272,        N282, Y305, R309, D319, Q320, Q359, K444 and/or G475, preferred        variants of SEQ ID NO: 2 include those having one of more of the        following substitutions: Q87E,R, Q98R, S125A, N128C, T131I,        T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R,        S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or        deletion in position R180 and/or S181 or of T182 and/or G183,        preferred amylase variants of SEQ ID NO: 2 include those having        the substitutions: N128C+K178L+T182G+Y305R+G475K;        N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;        S125A+N128C+K178L+T182G+Y305R+G475K; or        S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein the        variants are C-terminally truncated and optionally further        comprises a substitution at position 243 and/or a deletion at        position 180 and/or position 181; or    -   h) amylases having SEQ ID NO: 12 in WO 01/66712 or a variant        having at least 90% sequence identity to SEQ ID NO: 12,        preferred amylase variants are those having a substitution, a        deletion or an insertion in one of more of the following        positions of SEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181,        G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302,        S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439,        R444, N445, K446, Q449, R458, N471 or N484, preferred amylases        include variants having a deletion of D183 and G184 and having        the substitutions R118K, N195F, R320K and/or R458K, and a        variant additionally having substitutions in one or more        position selected from the group: M9, G149, G182, G186, M202,        T257, Y295, N299, M323, E345 and A339, most preferred a variant        that has all these substitutions.-   Paragraph 23. The composition according to any of paragraphs 18 to    22, further comprising a lipase, wherein the lipase is selected from    lipase from Thermomyces, e.g. from T. lanuginosus as described in EP    258068 and EP 305216.-   Paragraph 24. The composition according to any of claims 17 to 23,    comprising:    -   a) at least 0.005 mg of active DNase variant according to any of        paragraphs 1 to 11 pr. Litre detergent, and optionally    -   b) 0 wt % to 60 wt % of at least one surfactant, and optionally    -   c) 0 wt % to 50 wt % of at least one builder.-   Paragraph 25. The composition according to paragraph 24, comprising    5 wt % to 60 wt % of at least one surfactant selected among    nonionic, anionic and/or amphoteric surfactants.-   Paragraph 26. The composition according to paragraph 25, wherein the    composition comprises an anionic surfactant preferably any of the    following: sulphate surfactants and in particular alkyl ether    sulphates, especially C-9-15 alcohol ethersulfates, C12-15 primary    alcohol ethoxylate, C8-C16 ester sulphates and C10-C14 ester    sulphates, such as mono dodecyl ester sulphates, including sulfates    and sulfonates, in particular, linear alkylbenzenesulfonates (LAS),    isomers of LAS, branched alkylbenzenesulfonates (BABS),    phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin    sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates),    hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such    as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS),    primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS    or FES, also known as alcohol ethoxysulfates or fatty alcohol ether    sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates    (PS), ester sulfonates, sulfonated fatty acid glycerol esters,    alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including    methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,    dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives    of amino acids, diesters and monoesters of sulfo-succinic acid or    salt of fatty acids (soap), and combinations thereof.-   Paragraph 27. The composition according to any of paragraphs 24 to    26, wherein the composition comprises at least one nonionic    surfactant including alcohol ethoxylates (AE or AEO), alcohol    propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty    acid alkyl esters, such as ethoxylated and/or propoxylated fatty    acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol    ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines,    fatty acid monoethanolamides (FAM), fatty acid diethanolamides    (FADA), ethoxylated fatty acid monoethanolamides (EFAM),    propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl    fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine    (glucamides, GA, or fatty acid glucamides, FAGA), as well as    products available under the trade names SPAN and TWEEN, and    combinations thereof. Commercially available nonionic surfactants    include Plurafac™, Lutensol™ and Pluronic™ from BASF, Dehypon™    series from Cognis and Genapol™ series from Clariant.-   Paragraph 28. The composition according to any of paragraphs 24 to    27, wherein the composition comprises at from 2 wt % to 50 wt % of    at least one builder.-   Paragraph 29. The composition according to paragraph 28, wherein at    least one builder is selected from: phosphates, sodium citrate    builders, sodium carbonate, sodium silicate, sodium aluminosilicate    (zeolite).-   Paragraph 30. The composition according to paragraph 29, wherein at    least one builder is selected from: zeolites, diphosphates    (pyrophosphates), triphosphates such as sodium triphosphate (STP or    STPP), carbonates such as sodium carbonate, soluble silicates such    as sodium metasilicate, layered silicates (e.g., SKS-6 from    Hoechst), and (carboxymethyl)inulin (CMI), and combinations thereof,    citrate, chelators such as aminocarboxylates, aminopolycarboxylates    and phosphonates, and alkyl- or alkenylsuccinic acid. Additional    specific examples include 2,2′,2″-nitrilotriacetic acid (NTA),    ethylenediaminetetraacetic acid (EDTA),    diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid    (IDS), ethylenediamine-N,N′-disuccinic acid (EDDS),    methylglycine-N,N-diacetic acid (MGDA), glutamic acid-N,N-diacetic    acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid,    N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic    acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid    (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic    acid (IDA), N-(sulfomethyl)aspartic acid (SMAS),    N-(2-sulfoethyl)-aspartic acid (SEAS), N-(sulfomethylglutamic acid    (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic    acid (MI DA), serine-N,N-diacetic acid (SEDA),    isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid    (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic    acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and    N′-(2-hydroxyethyl)ethylenediamine-N,N,N′-triacetic acid (HEDTA),    diethanolglycine (DEG), and combinations and salts thereof,    phosphonates including 1-hydroxyethane-1,1-diphosphonic acid (HEDP),    ethylenediaminetetrakis (methylenephosphonicacid) (EDTMPA),    diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or    DTPMPA or DTPMP), nitrilotris(methylenephosphonic acid) (ATMP or    NTMP), 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC) or    hexamethylenediaminetetrakis(methylenephosphonic acid) (HDTMP).-   Paragraph 31. A liquid laundry composition comprising:    -   a) at least 0.005 mg of DNase variant according to any of        paragraphs 1 to 11 per Litre of composition, and optionally    -   b) 0% to 30% by weight of at least one surfactant wherein the at        least one surfactant is selected from LAS, AEOS and/or SLES, and        optionally    -   c) 0% to 50% by weight of at least one builder selected from        citrate and/or methylglycine-N,N-diacetic acid (MGDA) and/or        glutamic-N,N-diacetic acid (GLDA) and/or salts thereof.-   Paragraph 32. A liquid laundry composition comprising:    -   a) at least 0.005 mg of DNase variant, according to any of        paragraphs 1 to 11, per Litre of composition, and optionally    -   b) 0% to 30% by weight of at least one surfactant wherein the at        least one surfactant is LAS, AEOS and/or SLES, and optionally    -   c) 0% to 50% by weight of at least one builder selected from        HEDP, DTMPA or DTPMPA.-   Paragraph 33. A composition according to any of paragraphs 27 to 32    comprising;    -   a) at least 0.005 mg of DNase variant according to any of        paragraphs 1 to 11 per gram of composition, optionally    -   b) 10-50 wt % builder and optionally    -   c) at least one bleach component-   Paragraph 34. A composition according to paragraph 33 comprising at    least one bleach component is selected from the sources of hydrogen    peroxide; sources of peracids; and bleach catalysts or boosters.-   Paragraph 35. A composition according to paragraph 34 comprising at    least one bleach component selected from one or more of the    following: tetraacetylethylenediamine (TAED), sodium    4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),    sodium 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), sodium    4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoic acid    (DOBA), sodium 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), acetyl    triethyl citrate (ATC), bleach catalysts including manganese    oxalate, manganese acetate, manganese-collagen, cobalt-amine    catalysts and manganese triazacyclononane (MnTACN) catalysts;    preferably complexes of manganese with    1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or    1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), such as    Me3-TACN, such as the dinuclear manganese complex    [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and    [2,2′,2″-nitrilotris(ethane-1,2-diylazanylylidene-κN-methanylylidene)triphenolato-κ3O]manganese(III).-   Paragraph 36. A composition according to any of paragraphs 17 to 35,    wherein the composition is a cleaning composition such as a    detergent composition, such as laundry or dish wash composition.-   Paragraph 37. A composition according to any of paragraphs 17 to 36,    wherein the composition is a liquid composition, comprising at least    20% by weight and up to 95% water, such as up to 70% water, up to    50% water, up to 40% water, up to 30% water, up to 20% water, up to    10% water, up to 5% water or up to 2% water.-   Paragraph 38. A composition according to any of paragraphs 17 to 37,    wherein the composition is granular, e.g. a powder laundry    composition or a dish wash composition.-   Paragraph 39. The use of a DNase variant according to any of    paragraphs 1 to 11 or a composition according to any of paragraphs    17 to 38 for deep cleaning of an item.-   Paragraph 40. Use of a DNase variant according to any of paragraphs    1 to 11 or a composition according to any of paragraphs 17 to 38 for    reduction and/prevention re-deposition.-   Paragraph 41. Use of a DNase variant according to any of paragraphs    1 to 11 or a composition according to any of paragraphs 17 to 38 for    reduction and/prevention of malodor.-   Paragraph 42. A method for laundering an item, wherein the method    comprises the steps of:    -   a. Exposing an item to a wash liquor comprising a DNase variant        according to any of paragraphs 1 to 11;    -   b. Completing at least one wash cycle; and    -   c. Optionally rinsing the item,        -   wherein the item is a textile.-   Paragraph 43. A method according to paragraph 42, wherein pH of the    liquid solution is in the range of 1 to 11, such as in the range 5.5    to 11, such as in the range of 7 to 9, in the range of 7 to 8 or in    the range of 7 to 8.5.-   Paragraph 44. A method according to paragraph 42 or 43, wherein the    wash liquor may have a temperature in the range of 5° C. to 95° C.,    or in the range of 10° C. to 80° C., in the range of 10° C. to 70°    C., in the range of 10° C. to 60° C., in the range of 10° C. to 50°    C., in the range of 15° C. to 40° C. or in the range of 20° C. to    30° C. In some aspects, the temperature of the wash liquor is 30° C.-   The invention is further described in the following paragraphs-   Paragraph 1 A DNase variant, comprising an alteration compared to    the polypeptide shown in SEQ ID NO: 1 at one or more positions    corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16,    17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38, 39, 40, 42, 49, 51,    52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78, 79, 80, 82, 83,    92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112, 116, 125, 126,    127, 130, 13, 135, 138, 139, 143, 144, 145, 147, 149, 152, 156, 157,    159, 160, 161, 162, 164, 166, 167, 168, 170, 171, 172, 173, 174,    175, 176, 177, 178, 179, 181 and 182 of SEQ ID NO: 1, wherein the    variant has a sequence identity to the polypeptide shown in SEQ ID    NO: 1 of at least 60% and the variant has DNase activity.-   Paragraph 2 The variant of paragraph 1, which comprises one or more    alterations compared to SEQ ID NO: 1, wherein the one or more    alteration(s) is selected from the group consisting of: T1I, T1L,    T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V,    S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T,    A10V, A10L, A10K, Q12S, Q12V, Q12E, S13D, S13Y, S13T, S13Q, S13F,    S13R, S13V, S13N, S13H, S13M, S13W, S13K, S13L, S13E, Q14M, Q14R,    N16S, A17C, A17V, A17E, A17T, A17S, T19K, T19N, T19L, T19S, T19I,    T19V, K21Q, K21E, K21M, T22P, T22A, T22V, T22D, T22R, T22K, T22M,    T22E, T22H, T22L, T22W, T22F, T22C, T22S, T22I, G24Y, S25P, S25T,    S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A, S27C, S27L, S27E,    G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, S39A, S39P,    S39Y, S39H, S39E, S39N, S39M, S39D, Q40V, S42G, S42C, S42D, S42L,    S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S, D56I,    D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T,    Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I,    S59H, N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W,    S68K, S68Y, S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H,    V76E, V76A, V76Y, V76N, V76M, V76R, V76I, V76F, T77N, T77Y, T77W,    T77R, F78L, F78I, F78H, F78V, F78Y, F78C, T79G, T79R, N80K, N80S,    S82L, S82E, S82K, S82R, S82H, D83C, D83F, D83L, L92T, A93G, E94N,    G99S, S101D, S101A, S102M, S102L, S102V, S102A, S102K, S102T, S102R,    T104S, T104P, T104A, T105V, T105I, K107L, K107C, K107R, K107H,    K107S, K107M, K107E, K107A, K107Q, K107D, Q109K, Q109R, Q109S,    A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E, S116K,    A125K, S126I, S126E, S126A, S126C, T127C, T127V, T127S, S130E,    G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A,    S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H,    A147R, A147K, A147Q, A147W, A147N, A147S, G149S, K152H, K152R,    S156C, S156G, S156K, S156R, S156T, S156A, T157S, Y159H, Y159F,    K160R, K160V, W161L, W161Y, G162Q, G162N, G162D, G162M, G162R,    G162A, G162S, G162E, G162L, G162K, G162V, G162H, S164R, S164H,    S164N, S164T, Q166D, S167M, S167L, S167F, S167W, S167E, S167A,    S167Y, S167H, S167C, S167I, S167Q, S167V, S167T, S168V, S168E,    S168D, S168L, K170S, K170L, K170F, K170R, T171D, T171E, T171N,    T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V, Q174L,    G175D, G175E, G175N, G175R, G175S, M176H, L177I, N178D, N178E,    N178T, N178S, N178A, S179E, S181R, S181E, S181D, S181I, S181F,    S181H, S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, S181A,    Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q,    Y182F, Y182L, Y182N, Y182I, Y182E, Y182T and Y182W, wherein the    variant has a sequence identity to the polypeptide shown in SEQ ID    NO: 1 of at least 60% and the variant has DNase activity.-   Paragraph 3 the variant of any of paragraphs 1-2, which has an    improved stability compared to the parent or compared to the DNase    having the polypeptide shown in SEQ ID NO: 1.-   Paragraph 4 the variant of any of paragraphs 1-3, wherein the    variant has at least 80%, at least 85%, at least 90%, at least 95%,    at least 96%, at least 97%, at least 98%, at least 99 sequence    identity to the polypeptide shown in SEQ ID NO: 1.-   Paragraph 5 the variant of any of paragraphs 1-4, wherein the total    number of alterations compared to SEQ ID NO: 1 is 1-20, e.g. 1-10    and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.-   Paragraph 6 a detergent composition comprising a variant according    to any of claims 1 to 5.-   Paragraph 7 the detergent composition of paragraph 6, further    comprising one or more detergent components.-   Paragraph 8 the detergent composition according to any of paragraphs    6-7, further comprising one or more additional enzymes selected from    the group comprising proteases, amylases, lipases, cutinases,    cellulases, endoglucanases, xyloglucanases, pectinases, pectin    lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and    mannanases, or any mixture thereof.-   Paragraph 9 the detergent composition according to any of paragraphs    6-8 in form of a bar, a homogenous tablet, a tablet having two or    more layers, a pouch having one or more compartments, a regular or    compact powder, a granule, a paste, a gel, or a regular, compact or    concentrated liquid.-   Paragraph 10 use of a detergent composition according to any of    paragraphs 6-9 in a cleaning process, such as laundry or hard    surface cleaning such as dish wash.-   Paragraph 11 a method for obtaining a DNase variant, comprising    introducing into a parent DNase an alteration at one or more    positions corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10, 12,    13, 14, 16, 17, 19, 21, 22, 24, 25, 27, 28, 29, 30, 32, 38, 39, 40,    42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78, 79,    80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112, 116,    125, 126, 127, 130, 13, 135, 138, 139, 143, 144, 145, 147, 149, 152,    156, 157, 159, 160, 161, 162, 164, 166, 167, 168, 170, 171, 172,    173, 174, 175, 176, 177, 178, 179, 181 and 182 of SEQ ID NO 1,    wherein the variant has an amino acid sequence which is at least 60%    identical to the polypeptide shown in SEQ ID NO 1, and recovering    the variant.-   Paragraph 12 the method of paragraph 11, wherein the variant    comprises two, three, four or five alterations compared to the    polypeptide shown in SEQ ID NO: 1.-   Paragraph 13 the method according to any of paragraph 11 or 12,    wherein the variant comprises one or more alterations selected from    the group consisting of: T1I, T1L, T1V, T1F, T1Y, T1M, T1E, G4N,    TSF, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P,    S9R, A10D, A10M, A10I, A10Q, A10T, A10V, A10L, A10K, Q12S, Q12V,    Q12E, S13D, S13Y, S13T, S13Q, S13F, S13R, S13V, S13N, S13H, S13M,    S13W, S13K, S13L, S13E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T,    A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, T22P,    T22A, T22V, T22D, T22R, T22K, T22M, T22E, T22H, T22L, T22W, T22F,    T22C, T22S, T22I, G24Y, S25P, S25T, S27N, S27I, S27M, S27D, S27T,    S27V, S27F, S27A, S27C, S27L, S27E, G28L, Y29W, S30K, S30D, S30H,    S30T, D32Q, I38V, I38M, S39A, S39P, S39Y, S39H, S39E, S39N, S39M,    S39D, Q40V, S42G, S42C, S42D, S42L, S42M, S42F, S42N, S42W, V49R,    L51I, K52I, K52Q, K52H, A55S, D56I, D56L, D56T, S57W, S57Y, S57F,    S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L,    S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L, T65I,    T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y, S68H, S68C, S68T,    S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M,    V76R, V76I, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H, F78V,    F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H,    D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M,    S102L, S102V, S102A, S102K, S102T, S102R, T104S, T104P, T104A,    T105V, T105I, K107L, K107C, K107R, K107H, K107S, K107M, K107E,    K107A, K107Q, K107D, Q109K, Q109R, Q109S, A112S, S116D, S116R,    S116Q, S116H, S116V, S116A, S116E, S116K, A125K, S126I, S126E,    S126A, S126C, T127C, T127V, T127S, S130E, G132R, D135R, T138Q,    W139R, R143E, R143K, S144Q, S144H, S144A, S144L, S144P, S144E,    S144K, G145V, G145E, G145D, G145A, A147H, A147R, A147K, A147Q,    A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G, S156K,    S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L,    W161Y, G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E,    G162L, G162K, G162V, G162H, S164R, S164H, S164N, S164T, Q166D,    S167M, S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C,    S167I, S167Q, S167V, S167T, S168V, S168E, S168D, S168L, K170S,    K170L, K170F, K170R, T171D, T171E, T171N, T171A, T171S, T171C,    A172G, A172S, L173T, L173A, L173V, Q174L, G175D, G175E, G175N,    G175R, G175S, M176H, L177I, N178D, N178E, N178T, N178S, N178A,    S179E, S181R, S181E, S181D, S181I, S181F, S181H, S181W, S181L,    S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C, Y182K,    Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N,    Y182I, Y182E, Y182T and Y182W compared to the polypeptide shown in    SEQ ID NO: 1.-   Paragraph 14 the method according to any of paragraphs 11-13,    wherein the DNase variant has at least 60%, such as at least 70%, at    least 80%, at least 90%, at least 95%, at least 96%, at least 97%,    at least 98%, or at least 99%, sequence identity to the polypeptide    shown in SEQ ID NO: 1.    Assays and Detergent Compositions    Composition of Model Detergent A (Liquid)

Ingredients: 12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG(monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soyasoap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodiumformiate, 0.2% DTMPA and 0.2% PCA (all percentages are w/w)

Composition of Model Detergent T (Powder)

Ingredients: 11% LAS, 2% AS/AEOS, 2% soap, 3% AEO, 15.15% sodiumcarbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelant, 2% sodiumcitrate, 1.65% AA/MA copolymer, 2.5% CMC and 0.5% SRP (all percentagesare w/w).

Composition of Model Detergent X (Powder)

Ingredients: 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodiumcarbonate, 1% sokalan, 35.5% sodium sulfate (all percentages are w/w).

Assay I: Testing of DNase Activity

DNase activity may be determined on DNase Test Agar with Methyl Green(BD, Franklin Lakes, N.J., USA), which was prepared according to themanual from supplier. Briefly, 21 g of agar was dissolved in 500 mlwater and then autoclaved for 15 min at 121° C. Autoclaved agar wastemperated to 48° C. in water bath, and 20 ml of agar was poured intopetridishes with and allowed to solidify by incubation o/n at roomtemperature. On solidified agar plates, 5 μl of enzyme solutions areadded and DNase activity is observed as colorless zones around thespotted enzyme solutions.

Methods

General methods of PCR, cloning, ligation nucleotides etc. arewell-known to a person skilled in the art and may for example be foundin in “Molecular cloning: A laboratory manual”, Sambrook et al. (1989),Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.); “Current protocols in Molecular Biology”, John Wiley and Sons,(1995); Harwood, C. R., and Cutting, S. M. (eds.); “DNA Cloning: APractical Approach, Volumes I and II”, D. N. Glover ed. (1985);“Oligonucleotide Synthesis”, M. J. Gait ed. (1984); “Nucleic AcidHybridization”, B. D. Hames & S. J. Higgins eds (1985); “A PracticalGuide To Molecular Cloning”, B. Perbal, (1984).

EXAMPLES Example 1: Construction of Variants by Site-DirectedMutagenesis

Site-directed variants were constructed of the Bacillus cibi DNase (SEQID NO: 1), comprising specific substitutions according to the invention.The variants were made by traditional cloning of DNA fragments (Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, 1989) using PCR together with properly designed mutagenicoligonucleotides that introduced the desired mutations in the resultingsequence.

Mutagenic oligos were designed corresponding to the DNA sequenceflanking the desired site(s) of mutation, separated by the DNA basepairs defining the insertions/deletions/substitutions, and purchasedfrom an oligo vendor such as Life Technologies. In order to test theDNase variants of the invention, the mutated DNA comprising a variant ofthe invention are integrated into a competent B. subtilis strain byhomologous recombination, fermented using standard protocols (yeastextract based media, 3-4 days, 30° C.), and screened by activity assay.

Expression and Purification

The constructed variants were plated on LB agar supplemented with 6ug/ml chloramphenicol and grown for 37° C. for one day. After growth,colonies were picked to individual wells of standard 96-well microtiterplates containing 200 ul TBgly broth supplemented with 6 ug/mlchloramphenicol and trace metals (50 mM FeCl3, 20 mM CaCl2, 10 mM MnCl2,10 mM ZnSO4, 2 mM CuCl2, and 2 mM NiCl2, (F. William Studier, “Proteinproduction by auto-induction in high-density shaking cultures”, ProteinExpression and Purification, 41 (2005) 207-234).

The wild type Bacillus cibi DNase, was also inoculated as reference infour wells on each microtiter plate. The microtiter plates were grownfor three days at 30° C. with shaking at 220 rpm. After growth, thesupernatants were screened for residual activity after stressing themfor 20 minutes at 48.5° C. in the presence of 96% (v/v) Model A.

Model Detergent A (Liquid)

12% LAS, 11% AEO Biosoft N25-7 (NI), 5% AEOS (SLES), 6% MPG(monopropylene glycol), 3% ethanol, 3% TEA, 2.75% coco soap, 2.75% soyasoap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodiumformate, 0.2% DTM PA and 0.2% PCA (all percentages are w/w), water isadded to 100%.

Example 2: Testing DNase Variants for Stability

Each supernatant sample were split into two identical samples bytransferring 10 ul supernatant to two 96-well standard microtiter plateseach containing 240 ul Model A. After shaking for five minutes at 7500rpm in a microtiter plate shaker, one microtiter plate was incubated atroom temperature (21° C.=reference condition) for 20 min and the othermicrotiter plate was incubated in a 96-well PCR block at the stresstemperature (48.5° C.=stress condition) also for 20 min. Both sets ofsamples (the reference condition and the stressed condition) werediluted 100-fold in dilution buffer (50 mM Tris, HCl, pH 7.5) beforeassaying the activity by usage of the DNAseAlert substrate™ solution(Integrated DNA Technologies/Belgium, part #11-04-02-04.

DNase Activity Assay II

For the DNase activity measurement, 10 ul 100-fold diluted referenceDNase sample and stress DNase sample were transferred to a new 384-wellmicrotiter plate and 40 ul DNAseAlert assay solution was added (50 mMTrisHCl, pH. 7.5, 5 mM MnCl2, 6 nM DNAseAlert substrate™). Thefluorescence (excitation 536 nm and emission 556 nm) was read each 90seconds for a total of 30 minutes. From the kinetic curves, the slope ofthe reference sample (activity under reference conditions) and thecorresponding stress sample (activity under stress conditions) wasdetermined by linear regression. The residual activity (RA) for eachDNase variant and the reference DNase (SEQ ID NO 1) was calculated as:

slope (stress sample)/slope(reference sample).

Half-Life Improvement Factor

The half-life improvement factor (HIF, T_(1/2) in minutes) for the DNasevariants and the DNase reference (SEQ ID NO 1) was calculated as: 20minutes×LN(0.5)/LN(RA). The half-life improvement factor (HIF) for thevariants was calculated as T_(1/2variant)/T_(1/2backbone). Improvedvariants were identified as variants having a half-life improvementfactor HIF larger than 1.0 (HIF reference (SEQ ID NO 1)=1.0).

The activity improvement factor (AIF) is calculated as the activity ofthe variants (slope at reference conditions) divided by the activity ofthe reference (SEQ ID NO 1; slope at reference conditions). Variantswith an improved AIF>1.0 was identified as having improved activitycompared to the corresponding reference (SEQ ID NO 1). The results areshown in Table 1 below.

TABLE 1 Half-life improvement factor for Bacillus cibi (SEQ ID NO 1)DNase variants Variant HIF SEQ ID NO 1 1 T1I 3.14 T1L 3.12 T1V 1.87 T1F1.34 T1Y 1.11 T1M 1.11 T1E 1.10 G4N 1.12 T5F 1.08 T5C 1.07 P6V 1.27 P6G1.09 S7D 1.19 S7T 1.09 K8V 1.23 S9K 1.16 S9Q 1.10 S9V 1.10 S9L 1.09 S9F1.08 S9P 1.07 S9R 1.05 A10D 1.31 A10M 1.15 A10I 1.11 A10Q 1.09 A10T 1.08A10V 1.08 A10L 1.08 A10K 1.06 Q12S 9.54 Q12V 3.84 Q12E 1.10 S13D 1.28S13Y 1.27 S13T 1.25 S13Q 1.20 S13F 1.19 S13R 1.19 S13V 1.15 S13N 1.15S13H 1.12 S13M 1.09 S13W 1.09 S13K 1.07 S13L 1.07 S13E 1.05 Q14M 1.15Q14R 1.06 N16S 1.09 A17C 3.16 A17V 1.28 A17E 1.19 A17T 1.15 A17S 1.10T19K 1.21 T19N 1.17 T19L 1.15 T19S 1.14 T19I 1.09 T19V 1.07 K21Q 1.43K21E 1.33 K21M 1.08 T22P 2.02 T22A 1.56 T22V 1.54 T22D 1.50 T22R 1.47T22K 1.40 T22M 1.39 T22E 1.33 T22H 1.30 T22L 1.30 T22W 1.22 T22F 1.22T22C 1.22 T22S 1.21 T22I 1.20 G24Y 1.25 S25P 1.27 S25T 1.21 S27N 1.22S27I 1.18 S27M 1.16 S27D 1.14 S27T 1.13 S27V 1.11 S27F 1.11 S27A 1.11S27C 1.08 S27L 1.07 S27E 1.06 G28L 1.06 Y29W 1.07 S30K 1.35 S30D 1.16S30H 1.14 S30T 1.11 D32Q 1.35 I38V 1.21 I38M 1.09 S39A 1.39 S39P 1.35S39Y 1.29 S39H 1.15 S39E 1.11 S39N 1.08 S39M 1.07 S39D 1.06 Q40V 2.22S42G 1.41 S42C 1.32 S42D 1.19 S42L 1.15 S42M 1.14 S42F 1.12 S42N 1.10S42W 1.06 V49R 1.17 L51I 1.10 K52I 2.18 K52Q 1.85 K52H 1.05 A55S 1.60D56I 1.56 D56L 1.17 D56T 1.14 S57W 4.61 S57Y 4.39 S57F 2.74 S57H 1.63S57C 1.53 S57P 1.50 S57V 1.26 S57R 1.15 S57T 1.10 Y58A 1.24 Y58T 1.16S59C 1.48 S59T 1.44 S59L 1.44 S59Q 1.43 S59V 1.38 S59K 1.22 S59R 1.16S59M 1.15 S59I 1.10 S59H 1.07 N61D 1.09 P63A 1.20 T65L 1.21 T65I 1.14T65V 1.10 T65R 1.07 T65K 1.06 S68V 1.74 S68I 1.31 S68W 1.28 S68K 1.13S68Y 1.12 S68H 1.10 S68C 1.10 S68T 1.10 S68L 1.09 V76G 4.38 V76L 1.89V76C 1.64 V76K 1.47 V76H 1.33 V76E 1.30 V76A 1.26 V76Y 1.25 V76N 1.20V76M 1.18 V76R 1.18 V76I 1.13 V76F 1.11 T77N 1.70 T77Y 1.33 T77W 1.18T77R 1.16 F78L 1.57 F78I 1.27 F78H 1.13 F78V 1.11 F78Y 1.07 F78C 1.06T79G 1.35 T79R 1.33 N80K 1.28 N80S 1.10 S82L 1.17 S82E 1.16 S82K 1.11S82R 1.10 S82H 1.07 D83C 1.15 D83F 1.11 D83L 1.07 L92T 1.14 A93G 1.82E94N 1.07 G99S 1.13 S101D 1.28 S101A 1.08 S102M 1.36 S102L 1.14 S102V1.13 S102A 1.12 S102K 1.11 S102T 1.07 S102R 1.06 T104S 1.10 T104P 1.10T104A 1.09 T105V 1.64 T105I 1.40 K107L 1.76 K107C 1.40 K107R 1.26 K107H1.25 K107S 1.19 K107M 1.18 K107E 1.18 K107A 1.15 K107Q 1.14 K107D 1.13Q109K 2.00 Q109R 1.41 Q109S 1.05 A112S 2.84 S116D 1.35 S116R 1.25 S116Q1.18 S116H 1.14 S116V 1.14 S116A 1.13 S116E 1.12 S116K 1.06 A125K 1.18S126I 1.64 S126E 1.21 S126A 1.12 S126C 1.10 T127C 1.38 T127V 1.38 T127S1.20 S130E 1.33 G132R 1.86 D135R 1.08 T138Q 1.06 W139R 1.16 R143E 1.43R143K 1.35 S144Q 1.32 S144H 1.27 S144A 1.26 S144L 1.17 S144P 1.16 S144E1.13 S144K 1.06 G145V 1.51 G145E 1.18 G145D 1.09 G145A 1.06 A147H 3.25A147R 2.69 A147K 2.13 A147Q 1.53 A147W 1.38 A147N 1.23 A147S 1.16 G149S1.15 K152H 1.23 K152R 1.13 S156C 1.68 S156G 1.14 S156K 1.11 S156R 1.10S156T 1.08 S156A 1.08 T157S 1.16 Y159H 1.18 Y159F 1.08 K160R 1.13 K160V1.08 W161L 1.16 W161Y 1.06 G162Q 1.65 G162N 1.40 G162D 1.32 G162M 1.19G162R 1.14 G162A 1.14 G162S 1.14 G162E 1.11 G162L 1.11 G162K 1.11 G162V1.09 G162H 1.08 S164R 1.26 S164H 1.19 S164N 1.13 S164T 1.06 Q166D 1.76S167M 1.54 S167L 1.48 S167F 1.43 S167W 1.34 S167E 1.27 S167A 1.24 S167Y1.19 S167H 1.19 S167C 1.14 S167I 1.13 S167Q 1.13 S167V 1.09 S167T 1.07S168V 1.86 S168E 1.35 S168D 1.27 S168L 1.10 K170S 3.08 K170L 1.33 K170F1.07 K170R 1.07 T171D 2.59 T171E 1.48 T171N 1.17 T171A 1.14 T171S 1.13T171C 1.11 A172G 1.37 A172S 1.09 L173T 1.73 L173A 1.51 L173V 1.13 Q174L1.22 G175D 2.14 G175E 1.72 G175N 1.43 G175R 1.12 G175S 1.10 M176H 1.06L177I 1.84 N178D 1.44 N178E 1.25 N178T 1.20 N178S 1.17 N178A 1.17 S179E1.10 S181R 1.44 S181E 1.21 S181D 1.19 S181I 1.15 S181F 1.15 S181H 1.15S181W 1.14 S181L 1.13 S181M 1.13 S181Y 1.11 S181Q 1.10 S181V 1.10 S181G1.10 S181A 1.07 Y182M 1.45 Y182C 1.23 Y182K 1.20 Y182G 1.19 Y182A 1.17Y182S 1.14 Y182V 1.13 Y182D 1.12 Y182Q 1.11 Y182F 1.09 Y182L 1.09 Y182N1.09 Y182I 1.08 Y182E 1.08 Y182T 1.07 Y182W 1.06

The invention claimed is:
 1. A variant of a DNase parent, wherein thevariant comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO:25) or ASXNRSKG (SEQ ID NO: 26), wherein the variant comprises one ormore substitution(s) compared to SEQ ID NO: 1, wherein the substitutionis selected from the group consisting of: T1I, T1V, T1Y, T1M, T1E, G4N,T5F, T5C, P6V, P6G, S7D, S7T, K8V, S9K, S9Q, S9V, S9L, S9F, S9P, S9R,A10D, A10M, A10I, A10Q, A10V, A10L, A10K, Q12S, Q12V, Q12E, Q14M, Q14R,N16S, A17C, A17V, A17E, A17T, T19K, T19L, T19S, T19I, T19V, K21E, K21M,G24Y, S25P, S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L, S27E,G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, Q40V, S42C, S42L,S42M, S42F, S42W, V49R, L51I, K52I, K52H, A55S, D56I, D56L, D56T, S57W,S57F, S57H, S57C, S57P, S57V, S57R, S57T, Y58A, Y58T, S59C, S59T, S59L,S59Q, S59V, S59K, S59R, S59M, S59I, S59H, N61D, P63A, T65L, T65I, T65V,T65R, T65K, S68V, S68I, S68W, S68Y, S68H, S68C, S68T, S68L, V76G, V76L,V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76F, T77N, T77Y,T77W, T77R, F78I, F78H, F78Y, F78C, T79G, T79R, N80K, S82L, S82E, S82K,S82R, S82H, D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A,S102M, S102L, S102V, S102A, S102K, S102T, S102R, T104P, T104A, T105V,T105I, K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107D,Q109R, Q109S, A112S, S116D, S116R, S116Q, S116H, S116V, S116A, S116E,S116K, A125K, S126I, S126E, S126A, S126C, T127C, T127V, S130E, G132R,D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144L, S144P, S144E,S144K, G145V, G145E, G145D, G145A, A147Q, A147W, A147S, G149S, K152H,K152R, S156C, S156G, S156K, S156R, S156T, S156A, T157S, Y159F, K160V,W161L, W161Y, G162Q, G162D, G162M, G162R, G162A, G162S, G162E, G162L,G162K, G162V, G162H, S164R, S164T, Q166D, S167M, S167L, S167F, S167W,S167E, S167A, S167Y, S167H, S167C, S1671, S167Q, S167V, S167T, S168V,S168E, S168D, S168L, K1705, K170L, K170F, K170R, T171D, T171E, T171A,T171C, A172G, A172S, L173T, L173A, L173V, Q174L, G175D, G175E, G175N,G175R, M176H, L177I, N178D, N178E, N178T, N178S, N178A, S179E, S181R,S181E, S181D, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q, S181G,S181A, Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q,Y182F, Y182L, Y182N, Y1821, Y182E, Y182T and Y182W, wherein the varianthas a sequence identity to the polypeptide shown in SEQ ID NO: 1 of atleast 80% and the variant has DNase activity.
 2. The variant of claim 1,which has an improved stability, measured as half-life improvementfactor, HIF, compared to the parent or compared to the DNase having thepolypeptide shown in SEQ ID NO:
 1. 3. The variant of claim 1, whereinthe variant has at least 85% but less than 100% sequence identity to thepolypeptide shown in SEQ ID NO:
 1. 4. The variant of claim 1, whereinthe total number of alterations compared to SEQ ID NO: 1 is 1-20alterations.
 5. A detergent composition comprising a variant of claim 1.6. The detergent composition of claim 5, further comprising one or moredetergent components.
 7. The detergent composition of claim 5, furthercomprising one or more additional enzymes selected from the groupcomprising proteases, amylases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidaes, haloperoxygenases, catalases and mannanases, or any mixturethereof.
 8. The detergent composition of claim 6, in form of a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid.
 9. A methodfor obtaining a DNase variant, comprising: a) introducing into a parentDNase an alteration at one or more positions corresponding to positions1, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 19, 21, 24, 25, 27, 28, 29, 30,32, 38, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77,78, 79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112,116, 125, 126, 127, 130, 132, 135, 138, 139, 143, 144, 145, 147, 149,152, 156, 157, 159, 160, 161, 162, 164, 166, 167, 168, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 181 and 182 of SEQ ID NO 1, whereinthe variant has DNase activity; and b) recovering the variant.
 10. Themethod of claim 9, wherein the variant comprises 1-20 alterationscompared to the polypeptide shown in SEQ ID NO:
 1. 11. The methodaccording to claim 9, wherein the alteration is a substitution andwherein the substitution is selected from the group consisting of: T1I,T1L, T1V, T1F, T1Y, T1M, T1E, G4N, T5F, T5C, P6V, P6G, S7D, S7T, K8V,S9K, S9Q, S9V, S9L, S9F, S9P, S9R, A10D, A10M, A10I, A10Q, A10T, A10V,A10L, A10K, Q12S, Q12V, Q12E, Q14M, Q14R, N16S, A17C, A17V, A17E, A17T,A17S, T19K, T19N, T19L, T19S, T19I, T19V, K21Q, K21E, K21M, G24Y, S25P,S25T, S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A, S27C, S27L, S27E,G28L, Y29W, S30K, S30D, S30H, S30T, D32Q, I38V, I38M, Q40V, S42G, S42C,S42D, S42L, S42M, S42F, S42N, S42W, V49R, L51I, K52I, K52Q, K52H, A55S,D56I, D56L, D56T, S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, S57T,Y58A, Y58T, S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, S59H,N61D, P63A, T65L, T65I, T65V, T65R, T65K, S68V, S68I, S68W, S68K, S68Y,S68H, S68C, S68T, S68L, V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y,V76N, V76M, V76R, V761, V76F, T77N, T77Y, T77W, T77R, F78L, F78I, F78H,F78V, F78Y, F78C, T79G, T79R, N80K, N80S, S82L, S82E, S82K, S82R, S82H,D83C, D83F, D83L, L92T, A93G, E94N, G99S, S101D, S101A, S102M, S102L,S102V, S102A, S102K, S102T, S102R, T104S, T104P, T104A, T105V, T1051,K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, K107D,Q109K, Q109R, Q109S, A1125, S116D, S116R, S116Q, S116H, S116V, S116A,S116E, S116K, A125K, S1261, S126E, S126A, S126C, T127C, T127V, T127S,S130E, G132R, D135R, T138Q, W139R, R143E, R143K, S144Q, S144H, S144A,S144L, S144P, S144E, S144K, G145V, G145E, G145D, G145A, A147H, A147R,A147K, A147Q, A147W, A147N, A147S, G149S, K152H, K152R, S156C, S156G,S156K, S156R, S156T, S156A, T157S, Y159H, Y159F, K160R, K160V, W161L,W161Y, G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L,G162K, G162V, G162H, S164R, S164H, S164N, S164T, Q166D, S167M, S167L,S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S1671, S167Q, S167V,S167T, S168V, S168E, S168D, S168L, K170S, K170L, K170F, K170R, T171D,T171E, T171N, T171A, T171S, T171C, A172G, A172S, L173T, L173A, L173V,Q174L, G175D, G175E, G175N, G175R, G175S, M176H, L1771, N178D, N178E,N178T, N178S, N178A, S179E, S181R, S181E, S181D, S1811, S181F, S181H,S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, S181A, Y182M, Y182C,Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N,Y1821, Y182E, Y182T and Y182W compared to the polypeptide shown in SEQID NO:
 1. 12. The method according to claim 9, wherein the DNase parentbelongs to the GYS-clade and comprises one or both motifs[D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26). 13.The method of claim 9, wherein the parent DNase is selected from thegroup of polypeptides: a) a polypeptide having at least 80% sequenceidentity to the polypeptide shown in SEQ ID NO: 1, b) a polypeptidehaving at least 80% sequence identity to the polypeptide shown in SEQ IDNO: 2, c) a polypeptide having at least 80% sequence identity to thepolypeptide shown in SEQ ID NO: 3, d) a polypeptide having at least 80%sequence identity to the polypeptide shown in SEQ ID NO: 4, e) apolypeptide having at least 80% sequence identity to the polypeptideshown in SEQ ID NO: 5, f) a polypeptide having at least 80% sequenceidentity to the polypeptide shown in SEQ ID NO: 6, g) a polypeptidehaving at least 80% sequence identity to the polypeptide shown in SEQ IDNO: 7, h) a polypeptide having at least 80% sequence identity to thepolypeptide shown in SEQ ID NO: 8, i) a polypeptide having at least 80%sequence identity to the polypeptide shown in SEQ ID NO: 9, j) apolypeptide having at least 80% sequence identity to the polypeptideshown in SEQ ID NO: 10, k) a polypeptide having at least 80% sequenceidentity to the polypeptide shown in SEQ ID NO: 11, l) a polypeptidehaving at least 80% sequence identity to the polypeptide shown in SEQ IDNO: 12, m) a polypeptide having at least 80% sequence identity to thepolypeptide shown in SEQ ID NO: 13, n) a polypeptide having at least 80%sequence identity to the polypeptide shown in SEQ ID NO: 14, o) apolypeptide having at least 80% sequence identity to the polypeptideshown in SEQ ID NO: 15, p) a polypeptide having at least 80% sequenceidentity to the polypeptide shown in SEQ ID NO: 16, q) a polypeptidehaving at least 80% sequence identity to the polypeptide shown in SEQ IDNO: 17, r) a polypeptide having at least 80% sequence identity to thepolypeptide shown in SEQ ID NO: 18, s) a polypeptide having at least 80%sequence identity to the polypeptide shown in SEQ ID NO: 19, t) apolypeptide having at least 80% sequence identity to the polypeptideshown in SEQ ID NO: 20, u) a polypeptide having at least 80% sequenceidentity to the polypeptide shown in SEQ ID NO: 21, v) a polypeptidehaving at least 80% sequence identity to the polypeptide shown in SEQ IDNO: 22, w) a polypeptide having at least 80% sequence identity to thepolypeptide shown in SEQ ID NO: 23, and x) a polypeptide having at least80% sequence identity to the polypeptide shown in SEQ ID NO:
 24. 14. Themethod of claim 9, wherein the parent DNase is obtained from theBacillus genus.
 15. The method according to claim 9, wherein the DNasevariant has at least 80% sequence identity to the polypeptide shown inSEQ ID NO:
 1. 16. A method, comprising, exposing a textile or hardsurface to the detergent composition of claim 5, completing at least onewash cycle, and optionally rinsing the textile or hard surface.